brito-et-al-2025-caspase-1-activation-drives-vascular-inflammatory-processes-and-hypoperfusion-in-intravascular-html.html

Caspase-1 Activation Drives Vascular Inflammatory Processes and Hypoperfusion in

Intravascular Hemolysis

Pamela L. Brito,

Lucas, F.S. Gushiken,

Erica M.F. Gotardo,

Vanessa F. Alberto,

Flavia C.

Leonardo,

Amanda Becerra,

Dario S. Zamboni,

Fernando F. Costa,

Nicola Conran

Hematology and Transfusion Center, University of Campinas - UNICAMP, Campinas. São

Paulo, Brazil. 13083-878.

Department of Cellular and Molecular Biology. School of Medicine of Ribeirao Preto.

University of Sao Paulo, SP, Brazil. 14049-900.

Running title:

Caspase-1-Mediated Vascular Responses to Hemolysis

Corresponding author:

Nicola Conran, Hematology Center, Rua Carlos Chagas 480, Cidade

Universitaria, Barao Geraldo, Campinas, São Paulo, 13083-878, Brazil. Tel: +55 19 3251 5533.

conran@unicamp.br

KEY WORDS:

Adhesion molecules, cytokines, intravascular hemolysis, models, NLRP3-

inflammasome, oxidative stress, sickle cell disease.

A preprint version of this manuscript is available on bioRxiv at:

https://doi.org/10.1101/2025.03.10.642471

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

Abstract

Intravascular hemolysis (IVH), a pathological process associated with various conditions, triggers

inflammatory responses, yet the key molecular drivers of these responses are poorly defined,

particularly within the vasculature. To explore the role of NLRP3 inflammasome- and caspase-1-

dependent pathways in IVH-induced vascular dysfunction, we used

in vivo

models of acute and

chronic IVH, alongside heme stimulation of endothelial cells, thereby isolating this disease

mechanism from its etiological causes. IVH induced rapid inflammatory responses in C57BL/6J

mice, including IL-1β release within 15 minutes, and NLRP3-dependent caspase-1 activation in

circulating leukocytes. Chronic IVH elevated hepatic NLRP3 protein expression and caspase-1

activity in monocyte-derived macrophages. In turn, acute IVH significantly impaired cutaneous

microvascular blood flow and perfusion, and triggered microvascular leukocyte recruitment,

both via caspase-1-dependent mechanisms. Consistently, caspase-1-knockout mice did not

demonstrate hypoperfusion following IVH and pharmacological inhibition of caspase-1 in sickle

cell disease mice, which display continuous moderate hemolysis, attenuated heme-induced

vaso-occlusion. IVH also upregulated leukocyte CD11b-integrin expression

in vivo

, while heme

simulation of endothelial cells promoted inflammatory mediator release and caspase-1

activation, potentially facilitating leukocyte recruitment. Although the leukocyte alarmin

S100A8, released during IVH, amplified IL-1β release in the presence of heme, reactive oxygen

species generation, rather than caspase-1 activation, was required for endothelial adhesion

molecule expression. Together, these findings identify caspase-1 activation as a key driver of

hemolysis-induced microvascular leukocyte recruitment and hypoperfusion, potentially

facilitating the progression of skin lesions and organ damage. Targeting caspase-dependent

pathways may be of therapeutic potential for limiting vascular inflammation and tissue injury in

IVH-associated conditions.

NEW & NOTEWORTHY

This study identifies caspase-1 as a driver of the microvascular leukocyte recruitment and

hypoperfusion that is induced by intravascular hemolysis (IVH). Using

in vivo

models and heme-

stimulated endothelial cells, we show that IVH rapidly induces caspase-1-dependent

endothelial-leukocyte recruitment, microvascular dysfunction, and also IL-1β release. Oxidative

stress promotes heme-induced endothelial caspase-1 activation and adhesion molecule

expression, potentially amplifying vascular dysfunction. These findings provide insight into IVH-

driven pathology in hemolytic disorders, including sickle cell disease.

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

INTRODUCTION

Hemolysis, or the destruction of red blood cells (RBCs), is a major pathological process that can

be classified into two primary types: extravascular hemolysis, occurring predominantly in the

spleen and the liver, via macrophage-mediated phagocytosis, and intravascular hemolysis (IVH),

where RBC destruction occurs directly within the bloodstream (1). In addition to causing anemia,

hemolysis is a critical disease mechanism that drives systemic inflammation, upregulates

coagulation pathways and induces endothelial dysfunction, hallmarks of several disorders with

associated hemolysis, such as sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria

(PNH), malaria and hemolytic transfusion reactions (1-3). While these conditions exhibit distinct

etiologies and clinical manifestations, they share common hemolysis-related complications,

including pulmonary hypertension, cutaneous leg ulcers and priapism (4-6).

Intravascular hemolysis, in particular, has significant systemic effects, due to the

uncontrolled release of hemoglobin (Hb), heme and other erythrocyte contents into the

circulation. Cell-free Hb binds nitric oxide (NO), impairing vasodilation, while arginine released

from RBCs reduces NO bioavailability, contributing to endothelial dysfunction and

vasoconstriction (7). Typically, this process is controlled by haptoglobin, hemopexin, and other

heme-binding proteins, which scavenge free Hb and heme. However, during sustained

hemolysis, these protective systems become saturated leading to several consequences,

including complement system activation and rapid inflammatory responses (1, 8-11). In

addition, free heme, derived from oxidized Hb, promotes oxidative stress and acts as a danger-

associated molecular pattern (DAMP) (12, 13) that, together with other RBC-derived DAMPs,

may trigger innate immune responses, via Toll-Like receptor (TLR) signaling and inflammasome

activation, resulting in the processing and release of pro-inflammatory cytokines, such as

interleukin (IL)-1β and IL-18, and neutrophil extracellular traps (NETs)(14-16). Although such

molecular mechanisms implicate a major role of IVH in inducing immune responses, the exact

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

mechanisms by which IVH exacerbates the pathologies of hemolytic diseases remain

incompletely understood.

We previously standardized two murine models of hemolysis that emulate moderate

IVH. The acute IVH model, induced by a single administration of sterile water, triggers hypertonic

RBC lysis and leads to vascular inflammation (17). A similar water-infusion protocol in dogs led

to rapid NO depletion and endothelial dysfunction (3). In mice, acute IVH induces inflammatory

responses that include microvascular leukocyte recruitment and release of the S100A8 alarmin

(17). In contrast, our murine chronic IVH model is induced by repetitive low doses of

phenylhydrazine (LDPHZ), causing continuous RBC lysis through membrane peroxidation (18,

19). This model results in mild anemia, as evidenced by reticulocytosis and sustained depletion

of haptoglobin and hemopexin, ultimately driving inflammatory processes (18). The

inflammatory state that is induced by continuous hemolysis is characterized not only by elevated

plasma levels of pro-inflammatory molecules, along with endothelial cell activation, but also by

pathological hepatic alterations, including increased hepatic iron deposition, sinusoid

congestion and inflammatory cell infiltration (18). These models, which mimic moderate

hemolytic processes, have proven to be effective tools to investigate the mechanism of IVH-

induced inflammation in hemolytic disorders, independently of the cause of this hemolysis.

Inflammation plays a pivotal role in driving vascular disturbances through a cascade of

processes that lead to leukocyte recruitment, endothelial activation, and microvascular

100

occlusion (20). Pro-inflammatory molecules, such as TNF-ɑ and heme, can enhance the

101

expression and activation of adhesion molecules on endothelial cells and leukocytes (21-23),

102

promoting leukocyte adhesion to the vascular wall (24). This response is further amplified by

103

leukocyte mobilization, the release of additional pro-inflammatory cytokines and the generation

104

of reactive oxygen species (ROS) by activated leukocytes (18, 25, 26). Together with NO

105

depletion, generating impaired vasodilation and platelet activation, these inflammatory

106

processes exacerbate vascular dysfunction (27, 28). As such, IVH-driven inflammation and

107

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

hemodynamic changes may play a critical role in the pathology of hemolytic disorders, where

108

their interplay may be central to vascular dysfunction and microvascular occlusion.

109

One key mechanism that contributes to innate immune responses is inflammasome

110

activation (29). The multiprotein intracellular inflammasome complexes assemble in immune

111

cells in response to danger signals, leading to the activation of caspase-1, a protease that cleaves

112

pro-IL-1β and pro-IL-18 into their active forms (30). Active caspase-1 also induces gasdermin-

113

mediated pyroptosis, a form of programmed cell death that amplifies intracellular content and

114

cytokine release (31). Subsequently, IL-1β stimulates endothelial adhesion molecule expression

115

and enhances the production of inflammatory mediators such as IL-6, IL-8 and monocyte

116

chemoattractant protein-1 (MCP-1), thereby contributing to leukocyte recruitment (32, 33).

117

Additionally, NLRP3 inflammasome activation has been reported in non-immune cell types

118

including the endothelial cells themselves and platelets (34, 35). Hemolytic products, such as

119

heme and oxidized Hb, are potent DAMPs that are known to trigger NLRP3 inflammasome

120

activation in lipopolysaccharide (LPS)-primed macrophages and endothelial cells, leading to IL-

121

1β maturation and release (29, 36).

122

We hypothesized that inflammasome activation plays a key role in rapid multicellular

123

vascular inflammatory responses to acute IVH, contributing to vascular inflammation and

124

potentially to hemodynamic alterations. Over time, persistent hemolytic processes may drive

125

some of the long-term vascular complications associated with chronic hemolytic diseases. To

126

address this, we employed our animal models of hemolysis to replicate moderate acute and

127

chronic hemolysis without interference from underlying pathologies, allowing us to directly

128

assess the impact of the hemolytic process in vascular dysfunction. Additionally, we used

in vitro

129

assays to explore mechanisms of heme-stimulated macrovascular and microvascular endothelial

130

activation.

131

METHODS

132

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

Animals and Ethics Information

133

C57BL/6J (RRID:IMSR_JAX:000664) and transgenic Townes SCD (RRID:IMSR_JAX:013071) mice,

134

maintained at the animal facility (CEMIB) of the University of Campinas (UNICAMP), were used

135

aged 4 to 5 months for the study; Townes mice were bred from breeders originally purchased

136

from The Jackson Laboratory (Maine, USA).

Casp1

−/−

knock-out mice (RRID:IMSR_JAX:032662,

137

kindly provided by Russell Vance)(37) were bred at the University of São Paulo, FMRP/USP, Brazil

138

and used at 10 to 12 weeks old. These

Casp1Δ10

mice carry a CRISPR/Cas9-generated 10 bp

139

deletion in exon 8 of the mouse

Casp1

gene and was backcrossed to C57BL/6J for 4 generations.

140

The animals were housed in microisolators until euthanasia, with food and water provided

141

libitum

. All procedures were conducted in compliance with national guidelines for animal

142

experimentation and the study was approved by the Ethics Committee on Animal Use of

143

UNICAMP under protocols 5144-1/2019, 5144-1(A)/2022, 6435-1(A)/2025, 6672-1(A)/2025. All

144

efforts were made to minimize animal suffering and to use the minimum number of animals

145

necessary to obtain reliable and replicable results. Animals were euthanized at the end of final

146

procedures by deep anesthesia (300 mg/kg ketamine and 30 mg/kg xylazine,

i.p

.). Protocols for

147

C57BL/6J mice and Townes mice employed male mice due to the necessity to perform intravital

148

microscopy of a surgical preparation of the cremaster muscle.

Casp1

−/−

mice, employed only for

149

Doppler laser flowmetry, were both male and female; no differences in flowmetry were

150

observed between the two sexes.

151

Acute and Chronic Intravascular Hemolysis Induction

152

Induction of hemolytic processes was performed as previously described (17, 18). For the

153

induction of acute IVH, isogenic C57BL6/J mice received a single hemolytic stimulus via the

i.v.

154

administration of 150 µL of sterile water. As controls, mice of the same strain received an

155

injection of sterile saline solution (0.9% NaCl). Experimental groups were defined based on the

156

duration of hemolysis, with sample collection performed at 15 minutes, 1 hour, 3 hours and 6

157

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

hours after water or saline administration. For the induction of chronic IVH, C57BL6/J mice

158

received intraperitoneal (

i.p

.) injections of a low dose of phenylhydrazine (10 mg/kg; PHZ, Sigma-

159

Aldrich) twice weekly for four weeks, totaling 8 administrations. Similarly, control animals not

160

submitted to hemolysis received

i.p.

sterile saline solution (0.9% NaCl) administrations. Mice

161

from all groups were euthanized 48 hours after the last PHZ administration for sample collection.

162

Animal Treatment Protocols

163

Animals were randomly allocated to experimental groups to reduce potential selection bias.

164

Prior to the induction of acute IVH or saline administration, mice were pretreated for 1 hour

165

with the NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg,

i.p

, Sigma-Aldrich), while control

166

groups received sterile phosphate-buffered saline (PBS). For caspase-1 inhibition, mice were

167

treated with AC-YVAD-cmk (8 mg/kg, Sigma-Aldrich) or 0.5% Dimethyl Sulfoxide vehicle (DMSO,

168

Sigma-Aldrich) in sterile PBS, via

i.p

. injection, at 1 hour before IVH or saline or heme

169

administration. For heme stimulation of Townes SCD mice, mice received 32 μM heme (i.v., in

170

10 mM NaOH/PBS vehicle), to induce vaso-occlusive processes at 60 min before intravital

171

microscopy.

172

Dosing for

in vivo

administration of YVAD and MCC950 is shown in Supplementary Figure

173

1. The selected doses reflect titration based on overall performance across experiments, with 8

174

mg/kg YVAD and 50 mg/kg MCC950 generally yielding the most consistent and effective

175

responses, though not necessarily the maximal effect in every individual assay. While group

176

allocation was known during treatment administration, experimenters performing animal

177

analyses were blinded for data acquisition, to mitigate bias.

178

Laser Doppler Flowmetry

179

Cutaneous microvascular blood velocity and perfusion were assessed in the depilated pelvic

180

region of anesthetized mice (80 mg/kg ketamine/ 10 mg/kg xylazine,

i.p

.) using the PeriFlux 6000

181

system (Perimed Instruments, Järfälla, Sweden). Blood cell concentration (CMBC), velocity, and

182

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

perfusion were assessed/calculated for 10 minutes at a depth of 0.3 to 0.5mm, using a Ø1.0 mm

183

diameter probe with a 780 nm laser (38). Baseline measurements were taken 30 minutes before

184

hemolysis induction, and at 15-30 minutes after acute hemolysis induction or saline

185

administration. Absolute values were normalized to each animal’s baseline condition.

186

Intravital Microscopy

187

After surgical externalization of the cremaster muscle (38) in anesthetized mice (ketamine/

188

xylazine

, i.p

.), animals were placed under a microscope (Zeiss Imager M.2, Carl Zeiss Microscopy,

189

Jena, Germany) for visualization of the microvasculature. Segments (100 µm in length) from 4-7

190

different venules, each with a diameter of 25–35 µm, were selected at random from the central

191

area of the mounted cremaster muscle; often all available venules of the required size were

192

filmed. Images were acquired using an Axiocam 506 color camera (Carl Zeiss Microscopy) and

193

the Zen 2 Pro software (Carl Zeiss Microscopy) using a water-immersive 63X objective under

194

brightfield illumination for a maximum total filming time of 20 minutes. One-minute videos (57

195

frames per second) were exported as .avi files for quantitative analysis of segments. Rolling

196

leukocytes were defined as those moving at a slower velocity than that of free-flowing

197

erythrocytes in a given 100-µm venule segment over the 1-minute interval. Adherent leukocytes

198

were defined as those remaining stationary for the full duration of the video within the venular

199

segment. Extravasated leukocytes were defined as those that had migrated out of the

200

vasculature and into the surrounding tissue (up to 25 µm from the analyzed venule segment on

201

each side) in the interstitial space outside the vessel (18).

202

Isolation of Hepatic Non-Parenchymal Cells

203

For non-parenchymal cell (NPC) isolation, mouse livers were perfused

in situ

with perfusion

204

solution (Liver Perfusion Medium, 37°C, Gibco™, MA, USA), after which the liver was gently

205

dissected and digested with buffer containing collagenase (Liver Digest Medium, Gibco) at 37°C

206

for 40 minutes, with occasional agitation. After filtering (100-μm nylon cell strainer),

207

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

homogenates were washed three times (PBS, 30 × g for 4 minutes at 25°C) for hepatocyte and

208

debris sedimentation. The supernatant was then collected and subjected to two consecutive

209

centrifugations (300 ×

, 10 minutes, 22°C) to sediment non-parenchymal cells (NPCs), including

210

leukocytes and macrophages (39). The collected NPC fraction was carefully resuspended in PBS,

211

and centrifuged at 1500 ×

for 20 minutes at 25°C. The resulting pellet was resuspended in

212

sterile RPMI medium.

213

Endothelial Cell Culture and Treatments

214

Human Umbilical Vein Endothelial Cells (HUVEC; RRID:CVCL_2959)

and

Human Microvascular

215

Endothelial Cells (HMEC-1; RRID:CVCL_0307) were acquired from the American Type Culture

216

Collection (ATCC®, VA, USA). HUVECs were cultured in modified Ham’s F-12 Kaighn’s medium

217

(F12K, Gibco) with 10% fetal bovine serum (FBS, Invitrogen, MA, USA), endothelial growth factor

218

(30 ng/mL, Calbiochem, CA, USA), sodium heparin (10 U/mL, Cristália, São Paulo, Brazil), and

219

antibiotics (streptomycin and penicillin). HMEC-1 were cultured in MCDB 131 medium (Gibco)

220

with 10% FBS, 10 ng/mL Epidermal Growth Factor (EGF, Thermofisher, MA, USA), 1 µg/mL

221

hydrocortisone (Sigma-Aldrich), 10 mM L-Glutamine (Gibco), and antibiotics. Both cell types

222

were maintained in a humidified atmosphere at 37°C with 5% CO₂, and used at 80-90%

223

confluence, and between 3 and 6 passages. Both cell lines were morphologically checked by

224

microscopy for a cobblestone appearance and dark nuclei, and phenotypically characterized by

225

flow cytometry to ensure that they had maintained their phenotypes. HUVEC cells were CD31

226

CD34

low

, CD45

, CD105

, CD133

CD144

low

, CD146

low

, while HMEC-1 cells were

227

CD31

, CD146

, CD105

, CD34

low

, CD45

, CD133

(40-42). For experiments, HUVEC or HMEC-1

228

were seeded at 2 × 10⁵ cells/mL per well in complete F12K or MCDB 131 medium supplemented

229

with 2% FBS, for 24 hours at 37°C, 5% CO₂. Cells were stimulated, or not, with heme (50 µM;

230

Frontier Scientific, UT, USA) for 3 hours at 37°C, 5% CO₂. For caspase-1 inhibition, cells were

231

pretreated for 1 hour with YVAD-cmk (50 µM, Sigma-Aldrich) or 0.25 % dimethyl sulfoxide

232

vehicle (DMSO, Sigma-Aldrich) before heme stimulation for an additional 3 hours. For

233

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

antioxidant treatment, cells were incubated with 1 mM α-tocopherol (DL-all-rac-α-tocopherol,

234

Vitamin E, Sigma-Aldrich), or 2% ultrapure ethanol vehicle, for 30 minutes prior to stimulation

235

with heme. For human recombinant S100A8 incubation (ProSpec-Tany TechnoGene, Ness-Ziona,

236

CD, Israel), cells were pre-incubated with 1 µg/ml protein for 3 hours before heme stimulation.

237

Flow Cytometry: Murine Cells

238

Cellular caspase-1 activation and cell phenotyping was determined in murine cells using flow

239

cytometry. Fifty μL of EDTA-coagulated whole blood (for neutrophil and monocyte analysis) and

240

NPC samples (1 × 10⁶ cells/mL) were pre-incubated with a blocking solution (Mouse BD Fc Block

241

purified anti-mouse CD16/CD32 – 1:50, BD Pharmingen) for 15 minutes at room temperature

242

before incubating with the intracellular staining probe, FAM-YVAD-FMK (FAM-FLICA Caspase-1,

243

ImmunoChemistry Technologies, Bloomington, MN, USA), and phenotyping antibodies (37°C, 30

244

minutes). After incubation, red blood cells were lysed (BD Pharm Lyse Buffer; 15 minutes at

245

room temperature). Immediately after lysis, cells were washed (Apoptosis Wash Buffer; FAM-

246

FLICA Caspase-1 (YVAD) Assay Kit) and resuspended in PBS containing fixative solution for

247

acquisition on a FACSCalibur flow cytometer (BD Biosciences, CA, USA; RRID:SCR_000401), with

248

data analyzed by FlowJo software v.10.5.3 (RRID:SCR_008520). Viable neutrophils (Ly6G

high

249

CD11b⁺), monocytes (Ly6C

high

CD43⁺), and liver macrophages (F4/80⁺ CD11b⁺) were iden ﬁed

250

after gating based on SSC/FSC parameters. Caspase-1 activation was expressed as the

251

percentage of phenotyped cells that were FLICA⁺ (5x10

events). All antibodies used in flow

252

cytometry are listed in Supplementary Table 1.

253

Flow Cytometry: Endothelial Cells

254

For analysis of adhesion molecules and caspase-1 activity, HUVEC and HMEC-1 (1 × 10

cells/mL)

255

were fixed with 1% paraformaldehyde, then dissociated with 0.25% Trypsin/ 0.53 mM EDTA

256

solution (37°C) prior to antibody and FAM-FLICA Caspase-1 probe labelling for 30 min, 37°C (see

257

Supplementary Table 1). For ROS measurement, unfixed cells were collected, washed with PBS,

258

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

and immediately incubated with the 2′,7′-Dichlorodihydrofluorescein diacetate probe (DCFH-

259

DA, Sigma-Aldrich; 10 µM, 30 minutes, 37°C). Event acquisition with a FACSCalibur (2 × 10⁴

260

events), and data were analyzed using FlowJo software v.10.5.3. The gating strategy used

261

SSC/FSC (side scatter/forward scatter) parameters to identify viable cells and the population of

262

interest.

263

Immunoassay Protein Measurements

264

Murine plasma samples were used for enzyme-linked immunosorbent assays (ELISA) to quantify

265

IL-1β (mouse IL-1β/IL-1F2 [high sensitivity] ELISA Kit, R&D Systems, Minneapolis, MN, USA), IL-

266

18 (mouse IL-18 ELISA Kit, MBL), IL-6 (mouse IL-6, Abcam), TNF-α (Mouse TNF-alpha Quantikine

267

[high sensitivity] ELISA Kit, R&D Systems), S100A8 protein (DuoSet Mouse S100A8, R&D

268

Systems), and haptoglobin (Abcam), following the manufacturers’ instructions. Total liver

269

protein extracts were used to quantify hepatic IL-1β using the commercial mouse IL-1β

270

SimpleStep ELISA kit (Abcam), according to the manufacturer’s instructions. HUVEC

271

supernatants were used for ELISA to quantify IL-1β (human IL-1β/IL-1F2 [high sensitivity] ELISA

272

Kit, R&D Systems), IL-6 (human IL-6 Quantikine ELISA Kit, R&D Systems), MCP-1 (human

273

CCL2/MCP-1 Quantikine ELISA Kit, R&D Systems), IL-8 (human CXCL8/IL-8 Quantikine ELISA Kit,

274

R&D Systems).

275

Plasma Hemoglobin and Heme Measurement

276

Plasma hemoglobin was determined according to the method of Fairbanks et al. (43);

277

hemoglobin levels are expressed as µM heme bound to hemoglobin. Total cell-free plasma heme

278

was measured using a commercial kit (QuantiChrom™ Heme Assay Kit BioAssay Systems, San

279

Francisco, CA), according to the manufacturer’s instructions.

280

Quantitative Real Time PCR (qPCR)

281

mRNA from HUVECs was isolated using the RNeasy Micro Kit (Qiagen, Germany), according to

282

the manufacturer’s instructions. cDNA synthesis was performed using the RevertAid H Minus

283

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and cDNA concentration measured

284

using a NanoDrop spectrophotometer (Thermo Fisher Scientific). All qPCR samples were run in

285

duplicate on the same plate in a total volume of 12 µL, containing 10 ng of cDNA, 6 µL of SYBR

286

Green Master Mix PCR (Applied Biosystems, CA, USA), and oligonucleotide primers for the genes

287

of interest (

CASP1

NLRP3

and

PYCARD

, designed by Primer-Express, Applied Biosystems, and

288

synthesized by IDT, Iowa, USA). For oligonucleotide primer sequences and concentrations, see

289

Supplementary Table 2. The reaction was performed using the 7500 Fast Dx Real-Time PCR

290

instrument and software (Applied Biosystems). To confirm the accuracy and reproducibility of

291

the qPCR, intra-assay precision was calculated using the equation E(-1/slope), and a dissociation

292

curve analysis was performed at the end of each run to detect nonspecific amplifications. Results

293

are expressed as arbitrary units (A.U) of gene expression and were normalized to β-actin and

294

GAPDH expression, calculated using the geNorm program (44) (RRID:SCR_006763).

295

Protein Extraction and Immunoblotting

296

Liver fragments were homogenized in lysis buffer, containing a protease inhibitor cocktail. Total

297

protein was determined using the Bradford method. Fifty μg of protein extract were loaded onto

298

a 4-20% polyacrylamide gel (SDS-PAGE, Premade MiniProtean gel, Bio-Rad) for Western blotting.

299

The following primary antibodies (diluted 1:10) were used: rat anti-murine NLRP3 monoclonal

300

antibody (MAB7578; R&D Systems), rabbit anti-IL-1β polyclonal antibody (ab9722; Abcam),

301

rabbit anti-caspase-1 polyclonal antibody (ab138483; Abcam), and mouse anti-GAPDH

302

monoclonal antibody (sc-365062; Santa Cruz). Secondary antibodies (diluted 1:1000) were anti-

303

rat IgG-HRP-conjugated (sc-2006; Santa Cruz), anti-rabbit IgG-HRP-conjugated (sc-2357; Santa

304

Cruz) and anti-mouse IgG-HRP-conjugated (sc-358914; Santa Cruz). Blots were developed using

305

the Pierce ECL chemiluminescence detection kit (Thermo Scientific). Images were visualized and

306

captured using a photo documentation system (UVISoft- UVIBand, UVITEC Cambridge ALLIANCE-

307

6.7); band intensity values were normalized to the optical densities of GAPDH bands, and

308

analyzed with ImageJ software (RRID:SCR_003070).

309

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

For HUVEC, cells (2x10

) were washed twice with ice-cold PBS and lysed in RIPA buffer

310

(50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate)

311

supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA).

312

Lysates were incubated on ice for 10 minutes with intermittent vortexing and then centrifuged

313

at 14,000 × g for 15 minutes, 4°C. The supernatant was collected, and total protein concentration

314

was determined using the Bradford assay. Culture supernatants were centrifuged at 14,000 × g

315

for 10 minutes, 4°C to remove cellular debris. Samples (lysates or clarified supernatants) were

316

mixed with 6× Laemmli sample buffer containing 20% β-mercapethanol and boiled at 95°C for 5

317

minutes. For Western blotting, equal amounts of protein (20–30 µg) were separated by 4-15%

318

polyacrylamide gel (SDS-PAGE; Premade MiniProtean gel, Bio-Rad) and transferred onto

319

nitrocellulose membranes (Millipore). Membranes were incubated overnight at 4°C with

320

primary antibodies against IL-1β (0.3ug/mL rabbit anti-IL-1β polyclonal antibody, ab9722,

321

Abcam) or Caspase-1 (1:500 mouse anti-caspase-1 monoclonal antibody, sc56036, Santa Cruz

322

Biotechnology). After washing, membranes were incubated with HRP-conjugated secondary

323

antibodies (1:1000) for 1 hour at room temperature. Mouse anti-β-actin monoclonal antibody

324

HRP-conjugated (sc-47778, Santa Cruz Biotechnology, was used as loading control. Protein

325

bands were visualized using ultra-sensitivity ECL (SuperSignal™ West Femto Maximum

326

Sensitivity Substrate, Thermo Scientific, USA) and imaged as previous described.

327

Statistical Analysis

328

Data are expressed as means and standard error of N animals or cell samples. For comparisons

329

between two groups, the Mann-Whitney test was used. For comparisons between three groups

330

or more, data normality was assessed, and either a One-way Analysis of Variance (ANOVA)

331

followed by Holm-Sidak’s multiple comparison test (for parametric samples) or Kruskal-Wallis

332

test followed by Dunn’s multiple-comparison test (for non-parametric samples) was employed

333

using the GraphPad Prism software (RRID:SCR_002798). For the animal experiments, minimum

334

group sizes (5/ group) were calculated based on our prior experimental data (17, 38) and the

335

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

method described by Lehr et al. (45), using a statistical power of 0.8 (80%), and effect size (d) of

336

0.3. A probability (P value) of 5% or less was considered significant.

337

RESULTS

338

A Single Acute Hemolytic Event Induces a Rapid Pro-Inflammatory Effector Response in Mice

339

An acute intravascular hemolytic event was induced in C57BL/6J mice by injecting them (i.v.)

340

with sterile water, according to a previously established protocol (46). A control group received

341

a single administration of sterile saline. Blood samples were collected at 15 min, 1 hour and 3

342

hours later (Figure 1). As previously described (46), this protocol rapidly increases plasma cell-

343

free hemoglobin and total plasma cell-free heme, confirming the occurrence of IVH within 15min

344

(Figure 1A and B). Plasma haptoglobin was also rapidly depleted within 1 hour (Figure 1C). In

345

association with these observations, acute IVH triggered prompt inflammatory protein release,

346

with plasma levels of TNF-α cytokine increasing at 3 hours (Figure 1D). Furthermore, circulating

347

levels of the leukocyte-derived S100A8, (Figure 1E) an alarmin that can participate in

348

inflammasome activation, increased within just 15 minutes of the hemolytic stimulus.

349

Inflammasome activation is necessary for the maturation of the IL-1β and IL-18 cytokines;

350

consistent with the hypothesis of inflammasome-mediated cytokine processing, plasma levels

351

of IL-1β augmented within just 15 min of IVH induction (Figure 1F), while IL-18 release was

352

slower, occurring at approximately 6 hours post-hemolysis (Figure 1H). Additionally, liver IL-1β

353

protein content presented a significant increase within the first hour post-hemolysis (Figure 1G).

354

Acute

in vivo

Intravascular Hemolysis induces NLRP3-Dependent Caspase-1 Activation in

355

Murine Leukocytes

356

Having observed rapid hemolysis-driven inflammasome-derived cytokine release, we compared

357

caspase activity in the neutrophils and monocytes of mice that had received a single

i.v.

358

administration of either sterile 0.9% NaCl (control mice) or sterile water (IVH) to induce IVH

359

(Figure 2, and Supplementary Figure 2). C57BL/6J mice were also pretreated (1 hour before

360

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

hemolysis) intraperitoneally with either the irreversible caspase-1 inhibitor, ac-YVAD-CMK, or

361

the selective NLRP3-inflammmasome inhibitor, MCC950, or their vehicles (

i.p.

DMSO or sterile

362

PBS, respectively). Caspase activity was determined in immunophenotyped neutrophils and

363

monocytes at 1 hour post hemolysis (or saline infusion) using the FAM-YVAD-FLICA-FITC probe,

364

with primary specificity for caspase-1, and flow cytometry.

365

Acute IVH significantly increased caspase activity in both the neutrophils

366

(Ly6G

high

CD11b

, Figure 2A, C) and monocytes (Ly6C

high

CD43

, Figure 2B, D) of mice. Importantly,

367

this effect was prevented by both YVAD (Figure 2A, B) and by MCC950 (Figure 2C, D). While the

368

FLICA probe can detect the activity of other caspases (albeit with lower affinity), the combined

369

inhibition by YVAD and MCC950 strongly suggests that acute IVH induces NLRP3-dependent and,

370

therefore, presumably caspase-1 dependent activity in murine leukocytes.

371

Rapid Microvascular Leukocyte Recruitment in Response to Acute Intravascular Hemolysis is

372

Partially Mediated by Caspase-1 Activity.

373

The acute inflammatory response that is triggered by IVH rapidly induces leukocyte recruitment

374

to the microvascular wall at 15-35 minutes post-hemolysis, significantly increasing the rolling

375

and adhesion of these cells at the vascular wall and also increasing extravasation (Figure 3D-F),

376

compared to saline-control mice. When mice were pretreated (1 hour,

i.p

.) with the caspase-1

377

inhibitor, YVAD, both the rolling and adhesion of leukocytes in response to hemolysis were

378

abrogated (Figure 3A, B), although leukocyte extravasation was not significantly modulated

379

(Figure 3C). In contrast, the decrease in hemolysis-induced leukocyte rolling and adhesion

380

observed following pre-administration of the NLRP3 inhibitor, MCC950, was not statistically

381

significant (Figure 3D-F).

382

In association with these findings, we found no significant effect of IVH on the expression

383

of leukocyte CD11b at 15 minutes post induction; however, by 1 hour, CD11b expression on

384

leukocytes was significantly augmented. Flow cytometry demonstrated that the percentage of

385

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

CD11b

high

neutrophils and monocytes of mice that received saline was 63.2±3.4 % (n=35), while

386

this percentage was 62.6±3.6 % (n=13) at 15 min and 77.0±3.2 % (n=22,

<0.008 compared to

387

saline) at 1-hour post-IVH induction.

388

Acute

in vivo

Intravascular Hemolysis Significantly Impairs Microcirculatory Blood Perfusion in

389

an NLRP3- and Caspase-1-Dependent Manner

390

Since acute IVH induces microvascular leukocyte recruitment, we investigated the impact of this

391

event on microvascular blood flow and perfusion. Laser Doppler flowmetry was used to monitor

392

the cutaneous blood flow and perfusion in C57BL/6 mice at 15-35 min minutes after the

393

induction, or not, of acute IVH (Figure 4). Mice were also pretreated 1 hour prior to hemolysis

394

induction with

i.p.

YVAD or MCC950, and vehicle control groups were given either DMSO or

395

sterile PBS, respectively.

396

The induction of acute IVH in mice induced a very rapid and significant hypoperfusion in

397

the skin of mice, decreasing both blood flow velocity (Figure 4A, C) and blood perfusion (Figure

398

4B, D). Importantly, both caspase-1 (Figure 4A, B) and NLRP3 (Figure 4C, D) inhibition were able

399

to partially or fully restore the cutaneous microvascular blood flow and perfusion in mice

400

subjected to acute IVH, implying a role for the inflammatory response and NLRP3-inflammasome

401

activation in the ischemia observed.

402

The role of caspase-1 in this process was further supported by results obtained in

Casp1

403

knockout mice. Although intravascular hemolysis induced a rapid increase in plasma cell-free

404

Hb at 15 minutes in these mice (Figure 4G), no alterations in cutaneous blood flow or

405

microvascular perfusion were observed (Figure 4E, F). At 1 hour post-IVH, quantification of FAM-

406

YVAD-FLICA-FITC positivity in neutrophils and monocytes confirmed the absence of caspase

407

activity, indicating that the leukocyte caspase activity observed in wild-type C57BL/6J mice

408

subjected to IVH is principally mediated by caspase-1 (Figure 4 H, I; Supplementary Figure 3).

409

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

Chronic Intravascular Hemolysis in Mice Induces an Hepatic Inflammatory Response and

410

Inflammasome-Derived Cytokine Release

411

Using a previously-established model (18), we induced long-term continuous IVH in C57BL/6

412

mice by treating them for 28 days with low-dose phenylhydrazine (10 mg/kg

i.p

., 2x per week).

413

Control mice received

i.p.

saline at the same administration frequency. At 48 h after the last

414

phenylhydrazine dose, mice submitted to chronic IVH demonstrated significantly elevated

415

circulating levels of both IL-1β and IL-18 (Figure 5A, B), with similar concentrations to those

416

observed after a single acute IVH event. Western blotting also identified an increase in NLRP3

417

protein in the livers of mice that underwent chronic IVH, compared to control mice (Figure 5C,

418

Supplementary Figure 4F), but no significant alterations in other inflammasome components or

419

precursor molecules were observed in these livers (pro-caspase-1, caspase-1-p20 or -p15

420

subunits, pro-IL-1β, mature IL-1β subunit) (Supplementary Figure 4).

421

Immunophenotyping by flow cytometry of murine liver demonstrated a significant

422

increase in the number of infiltrated F4/80

CD11b

cells in mice subjected to chronic hemolysis,

423

compared to control mice (Figure 5D). Furthermore, these F4/80

CD11b

cells

displayed a higher

424

caspase activity in the hemolytic mice, as demonstrated by FLICA binding and flow cytometry

425

(Figure 5E, F).

426

Heme-Induced Microvascular Leukocyte Recruitment in a Murine Model of Chronic Hemolytic

427

Anemia is Partially Mediated by Caspase-1 Activity

428

To explore a potential role for caspase-1 activation in leukocyte recruitment in mice with chronic

429

hemolysis associated with disease etiology, we induced vaso-occlusive-like processes in Townes

430

transgenic mice with SCD using heme administration. The administration of heme (32 μM,

i.v

431

to SCD mice induced leukocyte recruitment to the microvascular wall at 60 min, significantly

432

increasing the rolling and adhesion of these cells, but not extravasation (Figure 5G-I), compared

433

to mice that received just vehicle (basal conditions). When SCD mice were pretreated (1 hour,

434

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

i.p

.) with the caspase-1 inhibitor, YVAD, both leukocyte rolling and adhesion were significantly

435

reduced (Figure 5G, H), although leukocyte extravasation was not significantly modulated

436

(Figure 5I).

437

Heme Induces Inflammatory Responses in Macro- and Microvascular Endothelial Cells

438

Considering the crucial roles of both leukocytes and the endothelium in vascular inflammation

439

and leukocyte recruitment, we examined the inflammatory responses of human umbilical vein

440

and microvascular endothelial cells (HUVEC and HMEC-1, respectively) to heme stimulation

, in

441

vitro

(Figure 6). Following incubation with heme (50 μM hemin) for 3 hours, both HUVEC and

442

HMEC-1 presented significant elevations in their surface expressions of the adhesion molecules

443

ICAM-1 (Figure 6A, D, G, H), VCAM-1 (Figure 6B, E, G, H) and E-selectin (Figure 6C, F, G, H). These

444

changes were associated with significant increases in the release of the inflammatory molecules,

445

IL-1β, IL-6, IL-8 and MCP-1 from heme-activated HUVEC (Figure 7A-D), highlighting the

446

inflammatory effect of the heme molecule on endothelial cells.

447

Priming of Heme-Stimulated Macrovascular Endothelial Cells Amplifies Cellular IL-1β release

448

Western blotting confirmed the presence of intracellular mature IL-1β and the p15 cleaved

449

caspase-1 fragment in lysed heme-stimulated cells, particularly when primed (Supplementary

450

Figure 5). However, IL-1β release from endothelial cells is lower than that observed for immune

451

cells and was therefore quantified in cell supernatants using a high-sensitivity ELISA. Notably,

452

priming of HUVEC with S100A8, which is released

in vivo

during IVH (Figure 1E), significantly

453

amplified the release of IL-1β from these cells (Figure 7A), but did not significantly alter the

454

effects of heme on IL-6, IL-8 or MCP-1 release from cells (Figure 7B-D). In comparison, IL-1β

455

release from heme-stimulated HUVEC after priming (3 hours) by the pathogen-associated

456

molecular pattern (PAMP), lipopolysaccharide (LPS; 100ng/ml), was similar to that of S100A8

457

primed cells, under our experimental conditions. For these experiments, supernatant IL-1β was:

458

<0.063 pg/ml (n=16 independent biological replicates) under basal conditions; 0.107±0.010

459

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

pg/ml (n=16) for heme-stimulated HUVEC; 0.173±0.003 pg/ml (n=14) for LPS-incubated cells;

460

0.384±0.031 pg/ml (n=7) for LPS-primed and heme-stimulated cells and 0.398±0.016 pg/ml

461

(n=16) for S100A8-primed and heme-stimulated cells (P=0.481, compared to LPS+Heme).

462

Heme-Induced Adhesion Molecule Expression by Endothelial Cells is Dependent on Reactive

463

Oxygen Species Generation and not Increased Caspase-1 Activity

464

To examine whether inflammasome activation participates in heme-induced inflammatory

465

responses in endothelial cells, we determined caspase activity in heme-activated HUVEC and

466

HMEC-1 using the FAM-FLICA probe and flow cytometry. Caspase activity was significantly

467

increased in both HUVEC and HMEC-1 cells following heme incubation, compared to control cells

468

(Figure 8A and 8H; Supplementary Figure 6). Abrogation of heme-induced caspase activity in

469

HUVEC by the YVAD inhibitor indicated a major role for caspase-1 in this caspase activity (Figure

470

8A) (dosing for YVAD in HUVEC is shown in Supplementary Figure 7). Importantly, qPCR revealed

471

that the expression of the gene encoding caspase-1 in HUVEC was also elevated in response to

472

heme stimulation (Figure 8B; gene expressions of other inflammasome components are shown

473

in Supplementary Figure 8). However, inhibition of caspase-1 in HUVEC using the YVAD peptide,

474

had no significant inhibitory effect on heme-activated adhesion molecule expression (Figure 8E-

475

G; Supplementary Figure 6E-H).

476

Of note, the ROS contents of both HUVEC and HMEC-1 were significantly amplified by

477

heme stimulation (Figure 7C and I), as demonstrated using the DCFDA probe and flow cytometry.

478

Importantly, neutralization of intracellular ROS generation by incubation with membrane-

479

permeable α-tocopherol prevented the increase in caspase activity in HUVEC (Figure 8D,

480

Supplementary Figure 6D). In turn, in contrast to the effects of caspase-1 inhibition, the

481

neutralization of ROS significantly abolished the inducing effects of heme on ICAM-1, VCAM-1,

482

and E-selectin expression on HUVEC (Figure 8E-G) and abrogated heme-induced E-selectin

483

expression on HMEC-1 (Figure 8J). These data suggest that the inflammatory effects of heme in

484

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

endothelial cells may, at least in part, be driven by oxidative stress, which contributes to

485

caspase-1 activation. However, the induction of adhesion molecule expression on these cells

486

does not appear to be necessarily mediated by inflammasome/caspase-1 activity

487

DISCUSSION

488

Although different hemolytic disorders have distinct clinical manifestations, some share certain

489

characteristics, including inflammatory and pro-thrombotic processes, as well as manifestations

490

such as pulmonary hypertension (1). Such manifestations are probably secondary to hemolysis

491

itself, specifically IVH, irrespective of the underlying cause of the disease and suggest that IVH

492

acts an independent disease mechanism (47, 48). While the association between inflammation

493

and hemolytic disorders, particularly SCD, is well documented (49-53), the specific role of the

494

inflammasome in driving this inflammatory response at the vascular level is unclear (11, 54).

495

Reports demonstrate a clear role for NLRP3-inflammasome formation in heme-induced primed

496

human macrophages (19, 54, 55), with roles for ROS generation and K

efflux (54, 56). Lethal

497

hemolysis in mice, induced by phenylhydrazine, appears to require toll-like receptor-4-

498

dependent cytokine production and mitochondrial ROS production, without necessarily the

499

need for the administration of a pathogen-associated molecular pattern (PAMP)(56) priming

500

molecule.

501

Given that the complications of hemolytic diseases often arise in a sterile environment,

502

i.e.

in the absence of infection, and therefore without PAMP-driven cellular priming, we explored

503

how IVH, as an isolated mechanism can trigger vascular inflammation (17, 18). Understanding

504

how IVH and its products impacts vascular physiology and hemodynamics, as well as cell function

505

in the vasculature, areas that have been underexplored to date, may reveal key mechanisms by

506

which IVH may contribute to the pathophysiology and thromboinflammatory complications of

507

hemolytic disorders. Using

in vivo

models of non-lethal acute and chronic IVH, we characterized

508

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

rapid and longer-term vascular inflammatory responses to IVH and explored a potential role for

509

NLRP3-inflammasome and caspase-1 activation in this mechanism.

510

As previously reported, our model of acute

in vivo

hemolysis induced a rapid and

511

moderate IVH in C57BL/6J mice, resulting in immediate elevations of plasma cell-free

512

hemoglobin and total plasma cell-free heme (17) that were comparable to those of mice with

513

SCD (17). These alterations were accompanied by a rapid depletion of circulating haptoglobin,

514

indicative of an acute hemolytic event. Circulating TNF-α, a potent pro-inflammatory cytokine

515

(57), was up-regulated within 3 hours of IVH, suggestive of a systemic inflammatory response.

516

Additionally, plasma S100A8, a leukocyte-derived putative biomarker for sepsis and a predictor

517

of mortality in heart failure patients (58, 59), increased significantly within just 15 minutes of

518

the acute hemolytic event, reflecting rapid leukocyte activation. Concomitantly, circulating IL-

519

1β levels were augmented at 15 minutes post-IVH, indicative of increased inflammasome

520

activity and cytokine processing.

521

Supporting a role for an inflammasome-derived inflammatory response to acute IVH,

522

liver IL-1β-cytokine content increased significantly within 1 hour in these mice, suggesting a

523

potential role for hepatic macrophages in amplifying this inflammation (60, 61). Consistent with

524

this hypothesis, mice subjected to continuous IVH for 4 weeks exhibited an increased frequency

525

of hepatic monocyte-derived (F4/80

CD11b

) macrophages and a subset of these cells displayed

526

augmented caspase activity, as previously reported in heme-induced LPS-primed macrophages

527

(54). Interestingly, although IL-18 also increased after acute IVH in mice, it was only detectable

528

in the circulation at 6 hours after the event. This delay may reflect the need for gene expression

529

upregulation to mediate increased pro-IL-18 production (62). However, sustained IVH over 4

530

weeks triggered a persistent rise in plasma IL-1β and IL-18, accompanied by the upregulation of

531

hepatic NLRP3 gene expression, highlighting a continuous role for the inflammasome during

532

hemolytic disorders.

533

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

Considering the rapid alterations and the apparent participation of leukocytes in the

534

hemolytic inflammatory response, alongside the concomitant upregulation of S100A8, a known

535

alarmin capable of priming the inflammasome (19), we monitored the activity of caspase-1, the

536

major effector of canonical inflammasomes, in murine neutrophils and monocytes following

537

acute IVH. Caspase-1 activity, measured using FAM-FLICA YVAD-FMK-probe binding (63), was

538

significantly elevated in both cell types at one hour after IVH. Furthermore, treatment

in vivo

539

with either the caspase-1 inhibitor, YVAD, or the NLRP3 inhibitor, MCC950, abrogated FLICA

540

fluorescence, suggesting that leukocyte NLRP3-inflammasome assembly contributes to the

541

elevation in circulating IL-1β following acute IVH. Although the inhibitor, AC-YVAD-CMK, may

542

exhibit cross reactivity with other caspases in humans (64), such off-target effects are unlikely

543

at the dose employed. Moreover, caspase-1-deficient mice did not display increased leukocyte

544

caspase activity following acute IVH, confirming caspase-1 activity specificity.

545

The NLRP3 inflammasome has been implicated in leukocyte recruitment mechanisms in

546

inflammation (65, 66), while heme has been shown to promote neutrophil adhesion,

in vitro

547

(23). Using intravascular microscopy to assess the microvascular circulation in the cremaster

548

muscle of mice, we confirmed the previous observation of the rapid effect of acute IVH on

549

microvascular leukocyte recruitment (17). To assess a role for NLRP3-inflammasome activity in

550

this event, we inhibited caspase-1 or NLRP3 activity in C57BL/6 mice before inducing IVH.

551

Caspase-1 inhibition significantly reduced IVH-induced microvascular leukocyte rolling and

552

adhesion, whereas NLRP3 inhibition only partially attenuated this response. The limited effect

553

of NLRP3 inhibition observed suggests that alternative inflammatory pathways also contribute

554

to the vascular inflammation observed. Notably, the NLRC5 immune sensor, which is

555

upregulated during PAMP and cytokine priming, can mediate heme-induced macrophage cell

556

death and interacts with NLRP12 to form a panoptosome complex (67). This study also reported

557

that NLRC5 plays a role in lethality in animal models primed with LPS and subjected to severe

558

hemolysis (67).

559

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

As a proof of concept, we also assessed the contribution of caspase-1 to heme-induced

560

leukocyte microvascular recruitment in a mouse model of SCD, a process known to trigger vaso-

561

occlusive-like processes (68). Caspase-1 inhibition abrogated this response, suggesting that

562

caspase-1-dependent pathways may play a role in driving vaso-occlusion in SCD. Indeed, IL-1β

563

blockade was recently shown to inhibit vaso-occlusion in SCD mice (38). However, it is unlikely

564

that IVH is the sole inflammatory driver of vaso-occlusion in SCD, as this mechanism involves

565

other factors such as physicochemical alterations in the RBCs and their membranes, and

566

hypoxia-related responses (69). This underscores the importance of isolating hemolysis as a

567

mechanism for study.

568

While we have previously reported the rapid effect of IVH on leukocyte recruitment in

569

the microcirculation (17), its impact on microcirculatory blood flow and tissue perfusion have

570

not been reported. Using laser Doppler flowmetry to monitor blood flow dynamics, we show

571

that acute IVH leads to significant and rapid reductions in blood flow velocity and perfusion

572

within the cutaneous microcirculation of mice. These findings suggest the onset of acute tissue

573

ischemic processes, potentially mediated by the observed vascular leukocyte recruitment to the

574

endothelium (70, 71). A role for NO consumption in this rapid hypoperfusion is plausible, given

575

the established effect of hemolysis and cell-free Hb release on vascular NO levels (72, 73).

576

Indeed, 6 hour-water infusion in dogs significantly elevates mean arterial pressure, and this

577

effect can be attenuated by NO inhalation (3); however, both the YVAD caspase inhibitor and

578

the MCC950 NLRP3 inhibitor restored cutaneous blood perfusion in mice subjected to IVH.

579

Consistent with this observation,

Casp1

⁻/⁻ knockout mice did not display the reduc ons in

580

cutaneous blood flow or perfusion that were observed in wild-type animals following IVH,

581

supporting a key role for caspase-1 in the ischemic microvascular response. Thus, while caspase-

582

dependent leukocyte recruitment induced by IVH may not depend entirely on NLRP3, NLRP3-

583

inflammasome-mediated responses significantly contribute to IVH-induced impairment of

584

microvascular hemodynamics. These differences may reflect distinct molecular mechanisms and

585

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

temporal dynamics governing leukocyte recruitment and migration, and consequent ischemic

586

processes. Importantly, impaired skin perfusion as a result of IVH may contribute to cutaneous

587

lesions, such as skin ulcers (74, 75)

a feature of some IVH-associated diseases (76, 77). Indeed,

588

we recently showed that acute IVH also disrupts neovascularization processes in mice (78).

589

As the identified roles for NLRP3-inflammasome and caspase-1 activation in IVH-induced

590

microvascular leukocyte recruitment and ischemia,

in vivo

, may involve endothelial cell

591

interactions, we explored the effects of heme on endothelial activation and inflammasome

592

activity,

in vitro

. Endothelial cells display NLRP3 inflammasome assembly under certain

593

inflammatory conditions (36, 79, 80), and this activity may contribute to their adhesive

594

interactions. The incubation of both macrovascular (HUVEC) and microvascular (HMEC-1)

595

endothelial cells with heme at a concentration similar to that found in the circulation of patients

596

with SCD and mice induced to acute hemolysis prompted significant upregulation of the surface

597

expressions of the adhesion molecules, ICAM-1, VCAM-1 and E-selection, which are all

598

important for mediating leukocyte interactions with the vascular endothelium (81). Additionally,

599

incubation with the heme molecule stimulated the production of inflammatory chemokines and

600

cytokines by endothelial cells, reaffirming its role in endothelial cell activation (14). Furthermore,

601

as most hemolytic diseases present sterile inflammation, yet inflammasome activation typically

602

requires priming for IL-1β maturation (82), we primed HUVECs with S100A8, an alarmin that we

603

found to be rapidly induced

in vivo

by the IVH process. S100A8 priming of HUVEC significantly

604

amplified the release of IL-1β from these cells, but did not alter the effects of heme on IL-6, IL-8

605

or MCP-1 release from cells. Findings indicate that sterile priming molecules, such as those

606

released by activated leukocytes, could participate in the caspase-1 driven effects of IVH in the

607

vasculature.

608

Notably, heme upregulated caspase-1 gene expression in macrovascular endothelial

609

cells and augmented caspase activity in both the HUVECs and HMEC-1 cells; this was

610

accompanied by the generation of ROS. Pre-incubation of HUVECs with the anti-oxidant, α-

611

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

tocopherol, reduced caspase activity similarly to caspase-1 inhibition, suggesting that heme-

612

induced ROS generation in response to heme may play an intracellular signaling role in

613

endothelial caspase-1 activation, consistent with the known role of ROS-triggered NLRP3

614

inflammasome activation in endothelial dysfunction (34) and reports of the priming of

615

inflammasomes and upregulation of NLRP3 expression by heme-induced ROS generation (82).

616

While caspase-1 inhibition by YVAD had no significant effect on heme-induced endothelial cell

617

adhesion molecule expression, α-tocopherol significantly reduced the surface upregulation of

618

ICAM-1, VCAM-1 and E-selectin on these HUVECs and partially abrogated E-selectin

619

upregulation on HMEC-1. These findings indicate that while ROS-induced caspase-1 activity is

620

associated with heme-induced endothelial activation, adhesion molecule upregulation appears

621

to be mediated directly by ROS signaling, independently of inflammasome pathways. NF-κB

622

pathway signaling is demonstrated to mediate heme-induced neutrophil adhesion molecule

623

expression (23), as well as ROS-induced endothelial cell activation in response to stimuli such as

624

oxidized low density lipoprotein (oxLDL) (83) and LPS (84).

625

Our findings suggest that caspase-1 activation and, consequently, rapid IL-1β processing

626

play a significant role in mediating inflammatory responses to IVH, contributing to major

627

microvascular alterations such as reduced tissue blood perfusion and leukocyte recruitment.

628

Moreover, NLRP3-inflammasome activation appears to drive this caspase-1-dependent

629

leukocyte activation, particularly in the context of hypoperfusion. While ROS generation appears

630

to primarily regulate vascular endothelial adhesive interactions in response to heme, caspase-1

631

activation may contribute to broader inflammatory consequences in these cells. Given the

632

significance of IVH in driving inflammatory responses across different disorders, further studies

633

are warranted to improve therapeutic strategies that reduce IVH and mitigate the effects of RBC

634

DAMP release. In addition to complement therapy (85), in use for certain hemolytic diseases,

635

promising approaches include hemopexin therapy, currently in clinical trials [NCT06699849].

636

Targeting inflammasome- and caspase-dependent pathways could further attenuate the

637

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

downstream effects of IVH, with potential for contributing to endothelial integrity, limiting

638

leukocyte activation, and reducing the ischemic injury associated with IVH.

639

DATA AVAILABILITY

640

Datasets will be made available from the corresponding author upon reasonable request.

641

SUPPLEMENTAL MATERIAL

642

Supplementary Tables 1-2 and Supplementary Figures 1-8:

643

doi.org/10.6084/m9.figshare.29825153.v1

644

Supplementary Videos 1-9: doi.org/10.6084/m9.figshare.29825156.v1

645

ACKNOWLEDGMENTS

646

The authors would like to thank Irene Santos, for assistance with flow cytometry analysis.

647

GRANTS

648

This study was supported by the São Paulo Research Foundation (FAPESP) [grant numbers

649

2018/08010-9 and 2019/18886-1, NC and FFC; and 2019/11342-6, DSZ]. PLB was recipient of a

650

fellowship from Coordination for the Improvement of Higher Education Personnel (CAPES),

651

Brazil. LSFG and EMFG receive post-doctoral fellowships from FAPESP (2021/11851-4 and

652

2023/10650-4). VF received an undergraduate fellowship from FAPESP [grant number

653

2023/09206-2].

654

DISCLOSURES

655

The authors declare that they have no conflicts of interest that are relevant to this study.

656

AUTHOR CONTRIBUTIONS

657

PLB and NC conceived and designed the research. PLB, LFSG, VF, FCL, and EMFG performed

658

experiments. PLB and NC analyzed data. AB and DSZ assisted with conceiving and designing the

659

research and data interpretation. PLB, FFC, and NC interpreted the results of the experiments.

660

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

PLB and NC drafted the manuscript. All authors edited and revised the manuscript and approved

661

the final version.

662

REFERENCES

Rother RP, Bell L, Hillmen P, and Gladwin MT

. The clinical sequelae of intravascular

663

hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease.

664

JAMA

293: 1653-1662, 2005. doi:10.1001/jama.293.13.1653

665

Conran N, and De Paula EV

. Thromboinflammatory mechanisms in sickle cell disease -

666

challenging the hemostatic balance.

Haematologica

105: 2380-2390, 2020.

667

doi:10.3324/haematol.2019.239343

668

Minneci PC, Deans KJ, Zhi H, Yuen PS, Star RA, Banks SM, Schechter AN, Natanson C,

669

Gladwin MT, and Solomon SB

. Hemolysis-associated endothelial dysfunction mediated by

670

accelerated NO inactivation by decompartmentalized oxyhemoglobin.

J Clin Invest

115:

671

3409-3417, 2005. doi:10.1172/JCI25040

672

Guillaud C, Loustau V, and Michel M

. Hemolytic anemia in adults: main causes and

673

diagnostic procedures.

Expert Rev Hematol

5: 229-241, 2012. doi:10.1586/ehm.12.3

674

Conran N

. Intravascular hemolysis: a disease mechanism not to be ignored.

Acta Haematol

675

132: 97-99, 2014. doi:10.1159/000356836

676

Rietschel RL, Lewis CW, Simmons RA, and Phyliky RL

. Skin lesions in paroxysmal nocturnal

677

hemoglobinuria.

Arch Dermatol

114: 560-563, 1978.

678

Gladwin MT, Kanias T, and Kim-Shapiro DB

. Hemolysis and cell-free hemoglobin drive an

679

intrinsic mechanism for human disease.

J Clin Invest

122: 1205-1208, 2012.

680

doi:10.1172/JCI62972

681

Jeney Vr, Balla Jz, Yachie A, Varga Z, Vercellotti GM, Eaton JW, and Balla Gr

. Pro-oxidant

682

and

cytotoxic

effects

circulating

heme.

Blood

100:

879-887,

2002.

683

doi:10.1182/blood.V100.3.879 %J Blood

684

Vallelian F, Buehler PW, and Schaer DJ

. Hemolysis, free hemoglobin toxicity, and scavenger

685

protein therapeutics.

Blood

140: 1837-1844, 2022. doi:10.1182/blood.2022015596

686

10.

Frimat M, Tabarin F, Dimitrov JD, Poitou C, Halbwachs-Mecarelli L, Fremeaux-Bacchi V,

687

and Roumenina LT

. Complement activation by heme as a secondary hit for atypical

688

hemolytic uremic syndrome.

Blood

122: 282-292, 2013. doi:10.1182/blood-2013-03-

689

489245

690

11.

Mendonca R, Silveira AA, and Conran N

. Red cell DAMPs and inflammation.

Inflamm Res

691

65: 665-678, 2016. doi:10.1007/s00011-016-0955-9

692

12.

Schaer DJ, Buehler PW, Alayash AI, Belcher JD, and Vercellotti GM

. Hemolysis and free

693

hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of

694

therapeutic proteins.

Blood

121: 1276-1284, 2013. doi:10.1182/blood-2012-11-451229

695

13.

Bozza MT, and Jeney V

. Pro-inflammatory Actions of Heme and Other Hemoglobin-Derived

696

DAMPs.

Front Immunol

11: 1323, 2020. doi:10.3389/fimmu.2020.01323

697

14.

Belcher JD, Chen C, Nguyen J, Milbauer L, Abdulla F, Alayash AI, Smith A, Nath KA, Hebbel

698

RP, and Vercellotti GM

. Heme triggers TLR4 signaling leading to endothelial cell activation

699

and vaso-occlusion in murine sickle cell disease.

Blood

123: 377-390, 2014.

700

doi:10.1182/blood-2013-04-495887

701

15.

Lee AH, Ledderose C, Li X, Slubowski CJ, Sueyoshi K, Staudenmaier L, Bao Y, Zhang J, and

702

Junger WG

. Adenosine Triphosphate Release is Required for Toll-Like Receptor-Induced

703

Monocyte/Macrophage Activation, Inflammasome Signaling, Interleukin-1β Production,

704

and the Host Immune Response to Infection.

Crit Care Med

46: e1183-e1189, 2018.

705

doi:10.1097/ccm.0000000000003446

706

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

16.

Chen G, Zhang D, Fuchs TA, Manwani D, Wagner DD, and Frenette PS

. Heme-induced

707

neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease.

Blood

708

123: 3818-3827, 2014. doi:10.1182/blood-2013-10-529982

709

17.

Almeida CB, Souza LE, Leonardo FC, Costa FT, Werneck CC, Covas DT, Costa FF, and Conran

710

. Acute hemolytic vascular inflammatory processes are prevented by nitric oxide

711

replacement or a single dose of hydroxyurea.

Blood

126: 711-720, 2015. doi:10.1182/blood-

712

2014-12-616250

713

18.

Gotardo EMF, Brito PL, Gushiken LFS, Chweih H, Leonardo FC, Costa FF, and Conran N

714

Molecular and cellular effects of in vivo chronic intravascular hemolysis and anti-

715

inflammatory therapeutic approaches.

Vascul Pharmacol

150: 107176, 2023.

716

doi:10.1016/j.vph.2023.107176

717

19.

Silveira AAA, Mahon OR, Cunningham CC, Corr EM, Mendonca R, Saad STO, Costa FF,

718

Dunne A, and Conran N

. S100A8 acts as an autocrine priming signal for heme-induced

719

human Mvarphi pro-inflammatory responses in hemolytic inflammation.

J Leukoc Biol

106:

720

35-43, 2019. doi:10.1002/JLB.3MIA1118-418RR

721

20.

Sprague AH, and Khalil RA

. Inflammatory cytokines in vascular dysfunction and vascular

722

disease.

Biochemical

Pharmacology

78:

539-552,

2009.

723

doi:https://doi.org/10.1016/j.bcp.2009.04.029

724

21.

Shao Y, Saredy J, Yang WY, Sun Y, Lu Y, Saaoud F, Drummer C, Johnson C, Xu K, Jiang X,

725

Wang H, and Yang X

. Vascular Endothelial Cells and Innate Immunity.

Arteriosclerosis,

726

Thrombosis,

and

Vascular

Biology

40:

e138-e152,

2020.

727

doi:doi:10.1161/ATVBAHA.120.314330

728

22.

Wagener FA, Feldman E, de Witte T, and Abraham NG

. Heme induces the expression of

729

adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells.

Proc Soc

730

Exp Biol Med

216: 456-463, 1997. doi:10.3181/00379727-216-44197

731

23.

Miguel LI, Leonardo FC, Torres LS, Garcia F, Mendonca R, Ferreira WA, Jr., Gotardo EMF,

732

Fabris FCZ, Brito PL, Costa FF, and Conran N

. Heme induces significant neutrophil adhesion

733

in vitro via an NFkappaB and reactive oxygen species-dependent pathway.

Mol Cell Biochem

734

476: 3963-3974, 2021. doi:10.1007/s11010-021-04210-5

735

24.

Turhan A, Weiss LA, Mohandas N, Coller BS, and Frenette PS

. Primary role for adherent

736

leukocytes in sickle cell vascular occlusion: a new paradigm.

Proc Natl Acad Sci U S A

99:

737

3047-3051, 2002. doi:10.1073/pnas.052522799

738

25.

Silva JAF, Gotardo EMF, Chweih H, Miguel LI, Ferreira WA, Jr., Hedlund B, Elford HL,

739

Leonardo FC, Costa FF, and Conran N

. Didox (3,4-dihydroxybenzohydroxamic acid) reduces

740

the vascular inflammation induced by acute intravascular hemolysis.

Blood Cells Mol Dis

81:

741

102404, 2020. doi:10.1016/j.bcmd.2020.102404

742

26.

Kubes P

. Polymorphonuclear leukocyte--endothelium interactions: a role for pro-

743

inflammatory and anti-inflammatory molecules.

Can J Physiol Pharmacol

71: 88-97, 1993.

744

doi:10.1139/y93-013

745

27.

Ho-Tin-Noé B, Boulaftali Y, and Camerer E

. Platelets and vascular integrity: how platelets

746

prevent bleeding in inflammation.

Blood

131: 277-288, 2018. doi:10.1182/blood-2017-06-

747

742676

748

28.

Loscalzo J

. Nitric oxide insufficiency, platelet activation, and arterial thrombosis.

Circ Res

749

88: 756-762, 2001. doi:10.1161/hh0801.089861

750

29.

Dutra FF, and Bozza MT

. Heme on innate immunity and inflammation.

Front Pharmacol

751

115, 2014. doi:10.3389/fphar.2014.00115

752

30.

Lamkanfi M, and Dixit VM

. Mechanisms and functions of inflammasomes.

Cell

157: 1013-

753

1022, 2014. doi:10.1016/j.cell.2014.04.007

754

31.

Jimenez Fernandez D, and Lamkanfi M

. Inflammatory caspases: key regulators of

755

inflammation and cell death.

Biol Chem

396: 193-203, 2015. doi:10.1515/hsz-2014-0253

756

32.

Dinarello CA

. Immunological and inflammatory functions of the interleukin-1 family.

Annu

757

Rev Immunol

27: 519-550, 2009. doi:10.1146/annurev.immunol.021908.132612

758

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

33.

Kaul DK, Thangaswamy S, Suzuka SM, Fabry ME, Wanderer AA, and Gram H

. Anti-

759

Interleukin-1β Antibody-Based Therapy Ameliorates Endothelial Activation and

760

Inflammation

Sickle

Mice.

Blood

118:

848-848,

2011.

761

doi:10.1182/blood.V118.21.848.848

762

34.

Bai B, Yang Y, Wang Q, Li M, Tian C, Liu Y, Aung LHH, Li PF, Yu T, and Chu XM

. NLRP3

763

inflammasome in endothelial dysfunction.

Cell Death Dis

11: 776, 2020.

764

doi:10.1038/s41419-020-02985-x

765

35.

Vogel S, Arora T, Wang X, Mendelsohn L, Nichols J, Allen D, Shet AS, Combs CA, Quezado

766

ZMN, and Thein SL

. The platelet NLRP3 inflammasome is upregulated in sickle cell disease

767

via HMGB1/TLR4 and Bruton tyrosine kinase.

Blood Adv

2: 2672-2680, 2018.

768

doi:10.1182/bloodadvances.2018021709

769

36.

Erdei J, Toth A, Balogh E, Nyakundi BB, Banyai E, Ryffel B, Paragh G, Cordero MD, and

770

Jeney V

. Induction of NLRP3 Inflammasome Activation by Heme in Human Endothelial Cells.

771

Oxid Med Cell Longev

2018: 4310816, 2018. doi:10.1155/2018/4310816

772

37.

Rauch I, Deets KA, Ji DX, von Moltke J, Tenthorey JL, Lee AY, Philip NH, Ayres JS, Brodsky

773

IE, Gronert K, and Vance RE

. NAIP-NLRC4 Inflammasomes Coordinate Intestinal Epithelial

774

Cell Expulsion with Eicosanoid and IL-18 Release via Activation of Caspase-1 and -8.

775

Immunity

46: 649-659, 2017. doi:10.1016/j.immuni.2017.03.016

776

38.

Gotardo EMF, Torres LS, Zaidan BC, Gushiken LFS, Brito PL, Leonardo FC, Pellizzon CH,

777

Millholland J, Agoulnik S, Kovarik J, Costa FF, and Conran N

. Targeting P-selectin and

778

interleukin-1beta in mice with sickle cell disease: effects on vaso-occlusion, liver injury and

779

organ

iron

deposition.

Haematologica

110:

725-738,

2025.

780

doi:10.3324/haematol.2024.286418

781

39.

Lambertucci F, Motino O, Perez-Lanzon M, Li S, Plantureux C, Pol J, Maiuri MC, Kroemer

782

G, and Martins I

. Isolation of Primary Mouse Hepatocytes and Non-Parenchymal Cells from

783

a Liver with Precancerous Lesions.

Methods Mol Biol

2769: 109-128, 2024.

784

doi:10.1007/978-1-0716-3694-7_9

785

40.

Garlanda C, and Dejana E

. Heterogeneity of endothelial cells. Specific markers.

Arterioscler

786

Thromb Vasc Biol

17: 1193-1202, 1997. doi:10.1161/01.atv.17.7.1193

787

41.

Timmermans F, Van Hauwermeiren F, De Smedt M, Raedt R, Plasschaert F, De Buyzere

788

ML, Gillebert TC, Plum J, and Vandekerckhove B

. Endothelial outgrowth cells are not

789

derived from CD133+ cells or CD45+ hematopoietic precursors.

Arterioscler Thromb Vasc

790

Biol

27: 1572-1579, 2007. doi:10.1161/ATVBAHA.107.144972

791

42.

Munoz-Vega M, Masso F, Paez A, Carreon-Torres E, Cabrera-Fuentes HA, Fragoso JM,

792

Perez-Hernandez N, Martinez LO, Najib S, Vargas-Alarcon G, and Perez-Mendez O

793

Characterization of immortalized human dermal microvascular endothelial cells (HMEC-1)

794

for the study of HDL functionality.

Lipids Health Dis

17: 44, 2018. doi:10.1186/s12944-018-

795

0695-7

796

43.

Fairbanks VF, Ziesmer SC, and O'Brien PC

. Methods for measuring plasma hemoglobin in

797

micromolar concentration compared.

Clin Chem

38: 132-140, 1992.

798

44.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, and Speleman

799

. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of

800

multiple internal control genes.

Genome Biology

3: research0034.0031, 2002.

801

doi:10.1186/gb-2002-3-7-research0034

802

45.

Lehr R

. Sixteen S-squared over D-squared: a relation for crude sample size estimates.

Stat

803

Med

11: 1099-1102, 1992. doi:10.1002/sim.4780110811

804

46.

Conran N, and Almeida CB

. Hemolytic vascular inflammation: an update.

Rev Bras Hematol

805

Hemoter

38: 55-57, 2016. doi:10.1016/j.bjhh.2015.10.004

806

47.

Morris CR

. Mechanisms of vasculopathy in sickle cell disease and thalassemia.

Hematology

807

Am Soc Hematol Educ Program

177-185, 2008. doi:10.1182/asheducation-2008.1.177

808

48.

Kato GJ, McGowan V, Machado RF, Little JA, Taylor Jt, Morris CR, Nichols JS, Wang X,

809

Poljakovic M, Morris SM, Jr., and Gladwin MT

. Lactate dehydrogenase as a biomarker of

810

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary

811

hypertension, and death in patients with sickle cell disease.

Blood

107: 2279-2285, 2006.

812

doi:10.1182/blood-2005-06-2373

813

49.

Conran N, and Belcher JD

. Inflammation in sickle cell disease.

Clin Hemorheol Microcirc

68:

814

263-299, 2018. doi:10.3233/CH-189012

815

50.

Kanavaki I, Makrythanasis P, Lazaropoulou C, Tsironi M, Kattamis A, Rombos I, and

816

Papassotiriou I

. Soluble endothelial adhesion molecules and inflammation markers in

817

patients with beta-thalassemia intermedia.

Blood Cells Mol Dis

43: 230-234, 2009.

818

doi:10.1016/j.bcmd.2009.06.002

819

51.

Caprari P, Profumo E, Massimi S, Buttari B, Rigano R, Regine V, Gabbianelli M, Rossi S,

820

Risoluti R, Materazzi S, Gullifa G, Maffei L, and Sorrentino F

. Hemorheological profiles and

821

chronic inflammation markers in transfusion-dependent and non-transfusion- dependent

822

thalassemia.

Front Mol Biosci

9: 1108896, 2022. doi:10.3389/fmolb.2022.1108896

823

52.

van Bijnen ST, Wouters D, van Mierlo GJ, Muus P, and Zeerleder S

. Neutrophil activation

824

and nucleosomes as markers of systemic inflammation in paroxysmal nocturnal

825

hemoglobinuria: effects of eculizumab.

J Thromb Haemost

13: 2004-2011, 2015.

826

doi:10.1111/jth.13125

827

53.

Parsanathan R, and Jain SK

. Glucose-6-phosphate dehydrogenase deficiency increases cell

828

adhesion molecules and activates human monocyte-endothelial cell adhesion: Protective

829

role of l-cysteine.

Arch Biochem Biophys

663: 11-21, 2019. doi:10.1016/j.abb.2018.12.023

830

54.

Dutra FF, Alves LS, Rodrigues D, Fernandez PL, de Oliveira RB, Golenbock DT, Zamboni DS,

831

and Bozza MT

. Hemolysis-induced lethality involves inflammasome activation by heme.

832

Proc Natl Acad Sci U S A

111: E4110-4118, 2014. doi:10.1073/pnas.1405023111

833

55.

Nyakundi BB, Toth A, Balogh E, Nagy B, Erdei J, Ryffel B, Paragh G, Cordero MD, and Jeney

834

. Oxidized hemoglobin forms contribute to NLRP3 inflammasome-driven IL-1beta

835

production upon intravascular hemolysis.

Biochim Biophys Acta Mol Basis Dis

1865: 464-

836

475, 2019. doi:10.1016/j.bbadis.2018.10.030

837

56.

Prestes EB, Alves LS, Rodrigues DAS, Dutra FF, Fernandez PL, Paiva CN, Kagan JC, and

838

Bozza MT

. Mitochondrial Reactive Oxygen Species Participate in Signaling Triggered by

839

Heme in Macrophages and upon Hemolysis.

J Immunol

205: 2795-2805, 2020.

840

doi:10.4049/jimmunol.1900886

841

57.

Zelova H, and Hosek J

. TNF-alpha signalling and inflammation: interactions between old

842

acquaintances.

Inflamm Res

62: 641-651, 2013. doi:10.1007/s00011-013-0633-0

843

58.

Wang Q, Long G, Luo H, Zhu X, Han Y, Shang Y, Zhang D, and Gong R

. S100A8/A9: An

844

emerging player in sepsis and sepsis-induced organ injury.

Biomed Pharmacother

168:

845

115674, 2023. doi:10.1016/j.biopha.2023.115674

846

59.

Bai B, Xu Y, and Chen H

. Pathogenic roles of neutrophil-derived alarmins (S100A8/A9) in

847

heart failure: From molecular mechanisms to therapeutic insights.

Br J Pharmacol

180: 573-

848

588, 2023. doi:10.1111/bph.15998

849

60.

Liu L, Lin L, Wang Y, Yan X, Li R, He M, Li H, Zhuo C, Li L, Zhang D, Wang X, Huang W, Li X,

850

Mao Y, Chen H, Wu S, Jiang W, and Zhu L

. L-AP Alleviates Liver Injury in Septic Mice by

851

Inhibiting

Macrophage

Activation

via

Suppressing

NF-kappaB

and

NLRP3

852

Inflammasome/Caspase-1 Signal Pathways.

J Agric Food Chem

72: 8460-8475, 2024.

853

doi:10.1021/acs.jafc.3c02781

854

61.

Bortolami M, Venturi C, Giacomelli L, Scalerta R, Bacchetti S, Marino F, Floreani A, Lise M,

855

Naccarato R, and Farinati F

. Cytokine, infiltrating macrophage and T cell-mediated

856

response to development of primary and secondary human liver cancer.

Dig Liver Dis

34:

857

794-801, 2002. doi:10.1016/s1590-8658(02)80073-1

858

62.

Pirhonen J

. Regulation of IL-18 expression in virus infection.

Scand J Immunol

53: 533-539,

859

2001. doi:10.1046/j.1365-3083.2001.00939.x

860

63.

Karmakar M, Katsnelson M, Malak HA, Greene NG, Howell SJ, Hise AG, Camilli A, Kadioglu

861

A, Dubyak GR, and Pearlman E

. Neutrophil IL-1beta processing induced by pneumolysin is

862

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on

863

K+ efflux.

J Immunol

194: 1763-1775, 2015. doi:10.4049/jimmunol.1401624

864

64.

Lipinska K, Malone KE, Moerland M, and Kluft C

. Applying caspase-1 inhibitors for

865

inflammasome assays in human whole blood.

J Immunol Methods

411: 66-69, 2014.

866

doi:10.1016/j.jim.2014.05.018

867

65.

Amaral FA, Costa VV, Tavares LD, Sachs D, Coelho FM, Fagundes CT, Soriani FM, Silveira

868

TN, Cunha LD, Zamboni DS, Quesniaux V, Peres RS, Cunha TM, Cunha FQ, Ryffel B, Souza

869

DG, and Teixeira MM

. NLRP3 inflammasome-mediated neutrophil recruitment and

870

hypernociception depend on leukotriene B(4) in a murine model of gout.

Arthritis Rheum

871

64: 474-484, 2012. doi:10.1002/art.33355

872

66.

Van Bruggen S, Jarrot PA, Thomas E, Sheehy CE, Silva CMS, Hsu AY, Cunin P, Nigrovic PA,

873

Gomes ER, Luo HR, Waterman CM, and Wagner DD

. NLRP3 is essential for neutrophil

874

polarization and chemotaxis in response to leukotriene B4 gradient.

Proc Natl Acad Sci U S

875

120: e2303814120, 2023. doi:10.1073/pnas.2303814120

876

67.

Sundaram B, Pandian N, Kim HJ, Abdelaal HM, Mall R, Indari O, Sarkar R, Tweedell RE,

877

Alonzo EQ, Klein J, Pruett-Miller SM, Vogel P, and Kanneganti TD

. NLRC5 senses NAD(+)

878

depletion, forming a PANoptosome and driving PANoptosis and inflammation.

Cell

187:

879

4061-4077 e4017, 2024. doi:10.1016/j.cell.2024.05.034

880

68.

Beckman JD, Abdullah F, Chen C, Kirchner R, Rivera-Rodriguez D, Kiser ZM, Nguyen A,

881

Zhang P, Nguyen J, Hebbel RP, Belcher JD, and Vercellotti GM

. Endothelial TLR4 Expression

882

Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Front Immunol

11: 613278, 2020.

883

doi:10.3389/fimmu.2020.613278

884

69.

Kato GJ, Piel FB, Reid CD, Gaston MH, Ohene-Frempong K, Krishnamurti L, Smith WR,

885

Panepinto JA, Weatherall DJ, Costa FF, and Vichinsky EP

. Sickle cell disease.

Nat Rev Dis

886

Primers

4: 18010, 2018. doi:10.1038/nrdp.2018.10

887

70.

Prestigiacomo CJ, Kim SC, Connolly ES, Jr., Liao H, Yan SF, and Pinsky DJ

. CD18-mediated

888

neutrophil recruitment contributes to the pathogenesis of reperfused but not

889

nonreperfused stroke.

Stroke

30: 1110-1117, 1999. doi:10.1161/01.str.30.5.1110

890

71.

Ritter LS, Orozco JA, Coull BM, McDonagh PF, and Rosenblum WI

. Leukocyte accumulation

891

and hemodynamic changes in the cerebral microcirculation during early reperfusion after

892

stroke.

Stroke

31: 1153-1161, 2000. doi:10.1161/01.str.31.5.1153

893

72.

Reiter CD, Wang X, Tanus-Santos JE, Hogg N, Cannon RO, 3rd, Schechter AN, and Gladwin

894

. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease.

Nat Med

895

1383-1389, 2002. doi:10.1038/nm1202-799

896

73.

Juttner AA, Danser AHJ, and Roks AJM

. Pharmacological developments in antihypertensive

897

treatment through nitric oxide-cGMP modulation.

Adv Pharmacol

94: 57-94, 2022.

898

doi:10.1016/bs.apha.2022.01.001

899

74.

Barbano B, Marra AM, Quarta S, Gigante A, Barilaro G, Gasperini ML, and Rosato E

. In

900

systemic sclerosis skin perfusion of hands is reduced and may predict the occurrence of

901

new digital ulcers.

Microvasc Res

110: 1-4, 2017. doi:10.1016/j.mvr.2016.11.003

902

75.

Minniti CP, Delaney KM, Gorbach AM, Xu D, Lee CC, Malik N, Koroulakis A, Antalek M,

903

Maivelett J, Peters-Lawrence M, Novelli EM, Lanzkron SM, Axelrod KC, and Kato GJ

904

Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia.

905

Am J Hematol

89: 1-6, 2014. doi:10.1002/ajh.23571

906

76.

Kato GJ, Gladwin MT, and Steinberg MH

. Deconstructing sickle cell disease: reappraisal of

907

the role of hemolysis in the development of clinical subphenotypes.

Blood Rev

21: 37-47,

908

2007. doi:10.1016/j.blre.2006.07.001

909

77.

Muller-Soyano A, Tovar de Roura E, Duke PR, De Acquatella GC, Arends T, Guinto E, and

910

Beutler

. Pyruvate kinase deficiency and leg ulcers.

Blood

47: 807-813, 1976.

911

78.

Gotardo EMF, Brito PL, Leonardo FC, Costa R, Soares R, Costa FF, and Conran N

. Acute

912

intravascular haemolysis rapidly shifts the balance of angiogenic factors and accelerates

913

neovascularization in vivo.

Br J Haematol

2025. doi:10.1111/bjh.20040

914

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

79.

Wan Z, Fan Y, Liu X, Xue J, Han Z, Zhu C, and Wang X

. NLRP3 inflammasome promotes

915

diabetes-induced endothelial inflammation and atherosclerosis.

Diabetes Metab Syndr

916

Obes

12: 1931-1942, 2019. doi:10.2147/DMSO.S222053

917

80.

Beltran-Garcia J, Osca-Verdegal R, Perez-Cremades D, Novella S, Hermenegildo C, Pallardo

918

FV, and Garcia-Gimenez JL

. Extracellular Histones Activate Endothelial NLRP3

919

Inflammasome and are Associated with a Severe Sepsis Phenotype.

J Inflamm Res

15: 4217-

920

4238, 2022. doi:10.2147/JIR.S363693

921

81.

Kluger MS

. Vascular endothelial cell adhesion and signaling during leukocyte recruitment.

922

Adv Dermatol

20: 163-201, 2004.

923

82.

Bolivar BE, Brown-Suedel AN, Rohrman BA, Charendoff CI, Yazdani V, Belcher JD,

924

Vercellotti GM, Flanagan JM, and Bouchier-Hayes L

. Noncanonical Roles of Caspase-4 and

925

Caspase-5 in Heme-Driven IL-1beta Release and Cell Death.

J Immunol

206: 1878-1889,

926

2021. doi:10.4049/jimmunol.2000226

927

83.

Yi L, Chen CY, Jin X, Zhang T, Zhou Y, Zhang QY, Zhu JD, and Mi MT

. Differential suppression

928

of intracellular reactive oxygen species-mediated signaling pathway in vascular endothelial

929

cells by several subclasses of flavonoids.

Biochimie

94: 2035-2044, 2012.

930

doi:10.1016/j.biochi.2012.05.027

931

84.

Liu X, Pan L, Wang X, Gong Q, and Zhu YZ

. Leonurine protects against tumor necrosis factor-

932

alpha-mediated inflammation in human umbilical vein endothelial cells.

Atherosclerosis

933

222: 34-42, 2012. doi:10.1016/j.atherosclerosis.2011.04.027

934

85.

Tampaki A, Gavriilaki E, Varelas C, Anagnostopoulos A, and Vlachaki E

. Complement in

935

sickle cell disease and targeted therapy: I know one thing, that I know nothing.

Blood Rev

936

48: 100805, 2021. doi:10.1016/j.blre.2021.100805

937

938

939

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

Legends to Figures

940

Figure 1

Acute intravascular hemolysis induces rapid inflammatory responses in C57BL/6J

941

Mice.

Mice received single intravenous administrations of sterile 0.9% NaCl (CON, data are from

942

6-19 mice per group) or sterile water (IVH, data are from 3-12 mice per group) and blood samples

943

were collected at 15 minutes, 1 hour, 3 hours, and 6 hours after administrations for analysis.

944

Saline control samples from different timepoints were pooled into a single group for analysis,

945

while IVH samples were analyzed separately for each timepoint. Plasma cell-free hemoglobin

946

(A) and cell-free heme (B) were measured using the Fairbanks and Quantichrom™ colorimetric

947

assays, respectively, and expressed as μM heme. Plasma Haptoglobin (C), TNF-α (D), S100A8 (E),

948

and IL-1β (F), as well as liver IL-1β (G) and plasma IL-18 (H) were determined by ELISA.

949

Figure 2. Acute intravascular hemolysis induces NLRP3-dependent caspase-1 activation in

950

murine leukocytes.

C57BL/6J mice were pretreated (

i.p

.) with YVAD (caspase-1 inhibitor, 8

951

mg/kg, A, B) or MCC950 (MCC; NLRP3 Inflammasome Inhibitor; 50 mg/kg, C, D) or with DMSO

952

vehicle (0.5% in sterile PBS, vehicle for YVAD), or with sterile PBS vehicle. One hour later, animals

953

received single intravenous administrations of either sterile 0.9% NaCl (CON, data are from 5

954

mice per group) or sterile water (IVH, data are from 5-7 mice per group) and blood samples were

955

collected 1 hour later for detection of active caspase-1 using FAM-YVAD-FLICA-FITC and flow

956

cytometry (50 000 cells analyzed per sample). Neutrophils (A, C) and monocytes (B, D) were

957

identified for analysis. (E) Representative gating strategy used for flow cytometry, cells with

958

intermediate FSC and SSC were first gated and then monocytes were identified as Ly6C

CD43

959

cells; for neutrophils, the granulocyte population (high FSC/SSC) was gated and neutrophils were

960

identified as Ly6G

CD11b

. FLICA positivity was assessed using dot plots, with thresholds defined

961

based on a non-probed blank control. Numbers represent % cells in the specified gate. Int:

962

intermediate; hi: high. Both data sets are for IVH/DMSO vehicle samples; representative FLICA

963

dot plots for each group are shown in Supplementary Figure 2.

964

Figure 3. Effects of caspase-1 and NLRP3 inhibition on leukocyte microvascular recruitment in

965

mice subjected to acute intravascular hemolysis.

C57BL/6J mice were pretreated (

i.p

., 1 hour)

966

with YVAD (8 mg/kg; A, B, C) or MCC950 (MCC; 50 mg/kg; D, E, F) or with DMSO vehicle (0.5% in

967

sterile PBS, vehicle for YVAD), or with sterile PBS (vehicle for MCC950). Intravital microscopy was

968

performed on the cremaster muscle at 15-35 minutes after intravenous administration of sterile

969

0.9% NaCl (CON, data are from 5 mice per group) or after the hemolytic stimulus (IVH, data are

970

from 5 mice per group). Results are expressed as means ± SEM, with values from each mouse

971

derived from the analysis of 4-7 venules per animal. Links for representative videos of each

972

experimental group are provided in Supplementary Material.

973

Figure 4. Acute intravascular hemolysis induces NLPR3- and caspase- dependent impairment

974

of cutaneous blood microcirculation hemodynamics in C57BL/6J mice, but not caspase-1-

975

deficient mice.

C57BL/6J mice were pretreated (

i.p

., 1 hour) with YVAD (8 mg/kg; A, B) or

976

MCC950 (MCC; 50 mg/kg; B, D) or with DMSO vehicle (0.5% in sterile PBS, vehicle for YVAD), or

977

with sterile PBS

(vehicle for MCC950)

. Non-invasive Laser Doppler flowmetry was used to

978

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

measure peripheral microcirculatory blood flow (A, C) and perfusion (B, D) in the skin of the hind

979

limb of anesthetized mice, at 15-30 minutes after intravenous administration of sterile 0.9%

980

NaCl (CON, data are from 6-7 mice per group) or sterile water (IHV, data are from 6-9 mice per

981

group). Flowmetry data (in arbitrary units) are normalized relative to baseline unstimulated

982

measurements taken from the same mice, at 30 minutes before treatments.

(E-G) Depict the

983

effects of the induction of acute intravascular hemolysis in caspase-1 deficient mice. Casp1

-/-

984

mice received single intravenous administrations of sterile 0.9% NaCl (CON, data are from 5

985

mice; 2F and 3M) or sterile water (IVH, data are from 7 mice; 3F and 4M) Laser Doppler

986

flowmetry demonstrated that the cutaneous microcirculatory blood flow (E) and perfusion (F)

987

in mice were unaltered in Casp1

-/-

mice submitted to IVH at 15-35 min post-hemolysis. Blood

988

samples collected at 15 minutes to quantify plasma cell-free hemoglobin (Hb) confirmed IVH.

989

Flow cytometry of caspase-1 activity, as demonstrated by FLICA positivity, in the neutrophils (H)

990

or monocytes (I) of the caspase-1 deficient mice subjected to IVH or not, at 1 hour (2M, 2F mice

991

in each group), as compared to aged-matched control C56BL/6J mice subjected to IVH (1 hour)

992

(4 mice). Representative dot plots for Casp1-/- mice are shown in Supplementary Figure 3.

993

Figure 5. Chronic in vivo intravascular hemolysis induces liver and macrophage inflammasome

994

activation, and caspase-1 activity may contribute to leukocyte recruitment in mice with sickle

995

cell disease.

C57BL/6J mice were repeatedly administered (

i.p

.) with

sterile 0.9% NaCl (CON,

996

2x/week) or low-dose phenylhydrazine (Chronic IVH, 10 mg/kg, 2x/week) for 4 weeks to induce

997

chronic IVH, or not. Peripheral blood and liver tissue samples were separated from the mice at

998

48 hours after the last administration. Plasma IL-1β (A; data are from 5-6 mice per group) and

999

IL-18 (B, data are from 7- 10 mice per group) were quantified by ELISA. The protein expression

1000

of the NLRP3 inflammasome subunit was quantitated by Western blotting in the liver samples

1001

(C, data are from 4 mice per group); expression was normalized by GAPDH expression in the

1002

same samples (Western blot membranes are displayed in Supplementary Figure 3). The

1003

frequency of F4/80

CD11b

macrophages in non-parenchymal cell (NPC) suspensions obtained

1004

from the liver samples was determined by flow cytometry (D, data are from 13-14 mice per

1005

group, 10 000 cells per sample). The percentage of these liver F4/80

CD11b

macrophages

1006

displaying caspase-1 activity was determined using the FAM-YVAD-FLICA-FITC probe and flow

1007

cytometry (E, data are from 6 mice per group, 10 000 cells per sample). (F) Gating strategy and

1008

representative dot plots showing the frequency of F4/80⁺ cells within the NPC popula on, and

1009

comparison of FLICA positivity between the F4/80⁺ cells of saline-treated and chronic IVH mice.

1010

(G-I) Townes SCD mice were pretreated (

i.p

., 1 hour) with YVAD (8 mg/kg, A, B) or with DMSO

1011

vehicle (0.5% in sterile PBS, vehicle for YVAD). Intravital microscopy was performed on the

1012

cremaster muscle at 60 minutes after intravenous administration of sterile 32 μM heme (data

1013

are from 6 mice per heme or heme/YVAD group) or sterile 10 mM NaOH (vehicle for heme;

1014

Basal; data are from 5 mice).

Results are expressed as means ± SEM, with values from each

1015

mouse derived from the analysis of 4-7 venules per animal.

Links for representative videos of

1016

each experimental group are provided in Supplementary Material.

1017

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.

Figure 6. Effects of heme on endothelial cell adhesion molecule expression,

in vitro

Human

1018

umbilical vein endothelial cells (HUVECs, A-C) or human microvascular endothelial cells-1

1019

(HMEC-1, D-F) were incubated under basal conditions (Basal), or with heme (50 μM) at 37°C, 5%

1020

CO₂ (3 hours). Cells (2 × 10⁵) were cultured in F12K medium supplemented with 2% fetal bovine

1021

serum (FBS). The percentage of cells expressing ICAM-1 (A, D), VCAM-1 (B, E), and E-selectin (C,

1022

F) was assessed by flow cytometry. Graphs depict the means ± SEM of data points from 7 to 15

1023

independent biological experiments, with 10 000 cells analyzed per sample. (G) Representative

1024

FSC/SSC dot plots for HUVEC and HMEC-1, and representative histograms depicting the

1025

comparison of CD54, CD106 or CD62E fluorescence for unstained, basal and heme-stimulated

1026

cells.

1027

Figure 7. Heme-induced release of endothelial inflammatory molecules, with or without

1028

S100A8 priming.

Human umbilical vein endothelial cells (HUVECs) were incubated under basal

1029

conditions (Basal), or with heme (50 μM) at 37°C, 5% CO₂ (3 hours) with or without

1030

preincubation (another 3 hours, plus co-incubation) with S100A8 (1 µg/ml). Cells (2 × 10⁵) were

1031

cultured in F12K medium supplemented with 2% fetal bovine serum (FBS). Concentrations of

1032

cyto/chemokines, IL-1β (A), IL-6 (B), IL-8 (C), and MCP-1 (D) in HUVEC supernatants were

1033

determined by high-sensitivity (IL-1β) or Quantikine ELISA. Each data point represents the

1034

average of duplicate measurements from 6-16 independent biological experiment; graphs show

1035

means ± SEM.

1036

Figure 8. Role of Caspase-1 activation and ROS generation in heme-induced endothelial cell

1037

activation, in vitro.

HUVEC (A-G) or HMEC-1 (H-J) were incubated under basal conditions (Basal),

1038

or with heme (50 μM) at 37°C, 5% CO₂ (3 hours). Cells (2 × 10⁵) were cultured in F12K medium

1039

supplemented with 2% FBS. Cells were also pretreated for 1h with YVAD (50 μM), or DMSO

1040

(YVAD vehicle, 0.25%), or for 30min with α-tocopherol (α-TP, 1 mM) or ethanol (ETOH, α-TP

1041

vehicle, 2%) before stimulating with 50 μM heme. (A and H) Quantification of endothelial

1042

caspase-1 activity using the FAM-YVAD-FLICA-FITC probe and flow cytometry. (B) Quantification

1043

of the expression of the gene encoding caspase-1 in HUVEC by qPCR. (C and I) Quantification of

1044

reactive oxygen species (ROS) generation in cells using the DCFDA/ H₂DCFDA probe and flow

1045

cytometry. Cellular expression of ICAM-1 (E), VCAM-1 (F), and E-selectin (CD62E) (G and J) was

1046

determined by flow cytometry. Graphs depict the means ± SEM of data points from 6 to 10

1047

independent biological experiments per group, with 10 000 cells analyzed per flow cytometry

1048

sample. Representative dot plots and histograms for each of the flow cytometry experiments

1049

are shown in Supplementary Figure 6.

1050

1051

Downloaded from journals.physiology.org/journal/ajpheart (2607:F720:1902:0011:0000:0000:0000:0129) on September 9, 2025.