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. Author manuscript; available in PMC: 2022 Dec 5.
Published in final edited form as: Microcirculation. 2022 Sep 22;29(8):e12782. doi: 10.1111/micc.12782

Histone-stimulated platelet adhesion to mouse cremaster venules in vivo is dependent on von Willebrand factor

Justin A Courson 1,2, Fong W Lam 1,3, Kimberly W Langlois 1,2, Rolando E Rumbaut 1,2,3,*
PMCID: PMC9720896  NIHMSID: NIHMS1849040  PMID: 36056797

Abstract

Objective:

Extracellular histones are known mediators of platelet activation, inflammation and thrombosis. Von Willebrand Factor (vWF) and Toll-like receptor 4 (TLR4) have been implicated in proinflammatory and prothrombotic histone responses. The objective of this study was to assess the role of vWF and TLR4 on histone-induced platelet adhesion in vivo.

Methods:

Intravital microscopy of the mouse cremaster microcirculation, in the presence of extracellular histones or saline control, was conducted in wild-type, vWF-deficient, and TLR4-deficient mice to assess histone-mediated platelet adhesion. Platelet counts following extracellular histone exposure were conducted. Platelets were isolated from vWF-deficient mice and littermates to assess the role of vWF on histone-induced platelet aggregation.

Results:

Histones promoted platelet adhesion to cremaster venules in vivo in wild-type animals, as well as in TLR4-deficient mice to a comparable degree. Histones did not lead to increased platelet adhesion in vWF-deficient mice, in contrast to littermate controls. In all genotypes, histones resulted in thrombocytopenia. Histone-induced platelet aggregation ex vivo was similar in vWF-deficient mice and littermate controls.

Conclusions:

Histone-induced platelet adhesion to microvessels in vivo is vWF-dependent and TLR4-independent. Platelet-derived vWF was not necessary for histone-induced platelet aggregation ex vivo. These data are consistent with the notion that endothelial vWF, rather than platelet vWF, mediates histone-induced platelet adhesion in vivo.

Keywords: von Willebrand Factor, vWF, histones, platelets, TLR4, Toll-like Receptor 4, cremaster, microvessel, aggregation, adherence

Introduction

Histones are small positively charged (pI ~11) proteins primarily found within the cell nucleus, where they serve to support chromatin structure and regulate gene expression [1,2]. Under normal conditions these highly cationic intra-nuclear proteins are contained within the cell; however, histones can be released into the extracellular space during cell death (necrosis and apoptosis) and in the specialized processes of NETosis (neutrophil extracellular trap formation) [3–7]. Histones are released passively during necrosis when intracellular content escapes as a result of plasma membrane rupture, and apoptotic cells can release histone via nucleosome formation and membrane blebbing [8,7]. During NETosis, neutrophils release histones and DNA, in a controlled manner, in an attempt to capture pathogens and combat infection [9]. When released into the extracellular space, histones have been recognized as mediators of platelet activation, inflammation and thrombosis. Consequently, extracellular histones have been implicated in a host of diseases and tissue injuries [3,10–12].

Extracellular histones primarily act as damage-associated molecular pattern molecules (DAMPs) [5]. As DAMPs, histones are linked to inflammation and thrombosis through their interactions with platelets, von Willebrand factor (vWF) and activation of toll-like receptors (TLRs) [5,13]. Anti-histone treatments have been shown to protect mice against trauma, stroke, coagulation, thrombosis, peritonitis, pancreatitis, ischemia/reperfusion injury, lethal endotoxemia and sepsis [14–21]. Furthermore, elevated levels of serum histones have been associated with the progression of cancer, inflammatory diseases, and autoimmune disorders [3,22–24]. Extracellular histones have been shown to have profound effects on platelet numbers and function, regardless of etiology. Extracellular histones injection alone in mice is enough to result in profound thrombocytopenia [25]. Histones have been shown to exacerbate trauma hemorrhage, and intravascular histone levels are elevated after severe non-thoracic blunt trauma and are positively correlated with severe complications and poorer prognosis [26,18]. Circulating histone levels are elevated in animal models of sterile liver, kidney, lung, and brain injury, suggesting an important role in the regulation and outcomes of sterile inflammation [27–30,16,23,19,20]. Extracellular histones may have both beneficial and detrimental effects in some diseases, such as sepsis. While histones have a bactericidal effect, extracellular histones also exacerbate microvascular dysfunction in sepsis and can result in death in animal models [31–33,21]. In addition, histones released via NETosis have been implicated in the progression of autoimmune and autoinflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, and small-vessel vasculitis [34–36].

Extracellular histones have a dramatic effect on platelets and can lead to platelet-mediated thrombosis in a number of conditions. Circulating levels of histones and nucleosomes have been associated with disease in patients with thrombotic microangiopathies as well as thrombocytopenia in critically ill patients [37,38]. These observations suggest that the interaction between platelets and extracellular histones may contribute to their pathological effects. In fact, depleting mice of platelets is sufficient to avoid death in response to exogenous histone administration [25]. As such, an understanding of the molecular players involved in this interaction is important. Evidence shows that histones can selectively bind to TLR2, −4, and −9 leading to the activation of signaling pathways (e.g., ERK, Akt, p38, and NF-κB) and the release of pro-inflammatory cytokines, which exacerbate tissue injury and the inflammatory response [27,16,12,20]. To this end, it has been shown that TLR4 deficiency in mice is enough to protect from extracellular histone-induced death [5]. This may result, in part, from the fact that histones can bind to TLR4 as well as platelet adhesion molecules, such as fibrinogen and vWF, making them particularly well equipped to stimulate platelet activation and aggregation [25,39–41].

A previous study from our lab showed that extracellular histones promote endothelial release of ultra-large vWF multimers ex vivo, and increased circulating vWF levels in histone-infused mice [13]. In addition, we have shown that histones induce platelet aggregation in human blood samples [42]. Histones can lead to the release of vWF from Weibel-Palade bodies in endothelial cells, as well as the release of vWF from platelet α-granules [10]. However, the role of vWF in histone-induced platelet adhesion in vivo is unclear. The purpose of this study was to define the role of vWF on platelet adhesion to microvessels in vivo, whether platelet-derived vWF contributes to histone-induced platelet aggregation, and whether TLR4 contributes to histone-induced platelet adhesion in vivo.

Materials & Methods

Animals

We studied 12–20-week-old male mice ~30 g in weight; all studies were in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and all protocols were approved by the Animal Care and Use Committee of Baylor College of Medicine and the Research & Development Committee of the Michael E. DeBakey VA Medical Center. Animals were either C57BL/6J (wild-type), C57BL/6(Cg)-TLR4tm1.1Karp (TLR4-deficient), or C57BL/6.129-St3gal4tm1.1Jxm (vWF-deficient) [43] mice and their littermate controls. Strains were originally ordered from the Jackson Laboratories, and TLR4- and vWF-deficient mice were genotyped using PCR analysis of tail clippings.

Animal preparation

The detailed methods for surgical preparation and intravital microscopy of cremaster muscle microcirculation were described in our prior publications [44,45]. Briefly, mice were anesthetized with sodium pentobarbital, maintained at 37°C using a homeothermic blanket, underwent tracheotomy to facilitate breathing, and placement of catheters in the internal jugular vein and carotid artery before exposing the cremaster muscle microvascular bed [46,47]. Throughout the experiment, the cremaster was superfused continuously with bicarbonate-buffered saline of the following composition (in mmol/l): 127 NaCl, 4.7 KCl, 2.0 CaCl2·2H2O, 1.2 MgSO4, 28.0 NaHCO3, and 5 glucose, at a rate of 5 ml/minute. The buffer was bubbled continuously with 95% N2-5% CO2 gas mixture to maintain a pH between 7.35 and 7.45. The temperature of the buffer at the tissue interface was maintained at 35°C.

Intravital microscopy of platelet adhesion to cremaster microvessels

After surgical preparation, animals were transferred to the stage of an upright intravital video-microscope (BX-50, Olympus) and allowed to equilibrate for 30 minutes. 6–10 animals were used per genotype/condition. 50 mg/kg of calf thymus histones (Worthington Biochemical; containing histone fractions H1, H2a, H2b, H3 & H4) were administered via the jugular catheter; histones were divided into two equal doses, given 15 minutes apart. This dosage was chosen as it has been shown to induce several characteristics of sepsis when administered in mice [21]. Imaging began 30 minutes after the last dose of histones was administered. The vasculature of each animal was carefully traced during the equilibration period in order to accurately identify 3rd order venules for video capture (Supplemental Figure 1). To avoid possible bias, in all cases the research staff member conducting intravital microscopy experiments was blinded as to the genotype or exogenous agent (histones vs. saline control). Visualization of individual microvessels was done with a 40x water immersion objective (numerical aperture 0.8). Immediately prior to recording, 10 μg of DyLight 488-conjugated anti-GPIbβ antibody (emfret Analytics) was injected intravenously to visualize circulating platelets. Upon identification of microvessels, 5 minutes of video were captured at 40x and subsequently assessed for platelet adhesion. Platelets were considered adherent if they were stationary on the microvascular walls for at least 5 seconds. Two separate 3rd order venules were studied per animal, and adherent platelet numbers averaged per animal, expressed as number of adherent platelets per unit surface area, assuming cylindrical vascular geometry.

Platelet counts in animals following intravital microscopy

Following intravital microscopy of cremaster microvessels, ~400 μl of blood were collected via the arterial catheter and placed in an EDTA-coated blood collection tube (Baxter, IL). Blood was then analyzed using a veterinary hematology analyzer (Heska Corporation; Loveland, Colorado) to determine platelet counts in whole blood.

Platelet Isolation for aggregation studies

To obtain washed platelets for aggregometry, animals were anesthetized using isoflurane and blood was collected from the inferior vena cava using a 20-gauge needle and syringe pre-coated with acid-citrate-dextrose (ACD; 100 mM trisodium citrate dihydrate, 111 mM dextrose, 71 mM citric acid) solution. Blood was collected in a syringe containing a 1:10 ratio of ACD to blood. Four animals per genotype/condition were used for this arm of the study. Collected blood was gently mixed with ACD solution before being transferred to a blood collection tube containing prostaglandin I2 (PGI2), and then diluted 1:1 with a 10x-diluted ACD buffer in PBS−/−. Samples were centrifuged at 68 RCF for 10 minutes without brakes. The supernatant was collected and centrifuged again to obtain platelet rich plasma. Washed platelets were then obtained by centrifuging the platelet rich plasma at 596 RCF for 10 minutes, removing the supernatant, and then resuspending the pellet in PBS−/− with 0.5 U/ml apyrase. The washed platelets were allowed to rest for 1 hour prior to platelet aggregometry.

Platelet aggregometry

Washed platelets were used to conduct light transmission aggregometry. Platelet concentrations were assessed using a Coulter Particle Counter (Beckman Coulter, CA), and samples adjusted to 2.5 × 108 platelets/ml using PBS−/− prior to platelet aggregometry. Studies were conducted on 225 μl of washed platelets. Immediately prior to aggregation, 4 μl of 100 mM CaCl2 and 1 μl of 100 mM MgCl2 were added to each sample. Platelet aggregation in response to calf thymus histone (CTH, 0.75 mg/ml), collagen (2 μg/ml), or saline control was measured in the washed platelets at 37°C and at 1200 rpm in a Bio/Data PAP-8 aggregometer (Bio/Data Corporation, PA).

Statistical Analysis

GraphPad Prism 9 (GraphPad Software, CA) was used for statistical analysis and data represented as the mean ± standard error of the mean. For platelet adhesion in vivo, and platelet counts in blood taken from mice following in vivo cremaster studies, comparisons by genotype within each test group (e.g., CTH vs. saline) were done with a Student’s t-test. For platelet aggregation studies, comparison of final platelet aggregation was done via a two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post-hoc test. A p-value of < 0.05 was considered statistically significant.

Results

Histone-enhanced microvascular platelet adhesion in vivo is independent of TLR4

Third-order venules were monitored for platelet adhesion in the presence or absence of extracellular histones. As shown in Figure 1, wild-type animals treated with calf thymus histones showed a significant increase in platelet adhesion when compared to animals treated with saline control (527 ± 83 vs. 148 ± 37 platelets/mm2, P < 0.05; supplemental video 1). In order to assess whether TLR4 was necessary for histone-induced platelet adhesion, TLR4 deficient mice (global knockouts) received either calf thymus histones or saline control (Figure 1). Similar to wild-type animals, TLR4-deficient mice treated with calf thymus histones showed a significant increase in platelet adherence when compared to animals treated with saline control (465 ± 119 vs 181 ± 45 platelets/mm2, P < 0.05). These data suggest that TLR4 is not necessary for histone-induced platelet adhesion to microvessels in vivo.

Figure 1. Extracellular histones stimulate platelet adhesion to microvessels in vivo in the absence of TLR4.

Figure 1

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Treating C57BL/6 (WT) mice (n = 6) with extracellular histones resulted in a significant increase in the number of adherent platelets in mouse cremaster venules compared to saline control. Each point represents an individual animal, with platelet adherence averaged over two separate venules per animal. Similarly, treating TLR4 deficient mice (n = 6) with extracellular histones resulted in a significant increase in platelet adhesion levels in the mouse cremaster venules compared to saline controls. These results suggest that the mechanism of action for histone-stimulated platelet adherence is TLR4 independent. * p < 0.05.

Histone-enhanced microvascular platelet adhesion in vivo is dependent on vWF

To determine whether vWF was necessary for histone-induced microvascular platelet adhesion in vivo, vWF-deficient mice (global knockouts) and their littermates were prepared for intravital imaging of the cremaster microvasculature, and either calf thymus histones or saline was administered. Third-order venules were then monitored for platelet adhesion. vWF-deficient mice showed no significant difference in platelet adhesion between mice treated with histones (179 ± 50 platelets/mm2) and mice treated with saline control (133 ± 40 platelets/mm2). In contrast, littermate control mice treated with extracellular histones showed a significant increase in adherent platelets when compared to littermates treated with saline control (385 ± 76 vs. 148 ± 43 platelets/mm2, P < 0.05) (Figure 2). These data suggest that in the absence of vWF signaling, intravascular histones do not stimulate platelet adherence to microvessels.

Figure 2. Extracellular histones stimulate platelet adhesion to microvessels in vivo in a vWF dependent manner.

Figure 2

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Treating vWF+/+ littermates (n = 9) with extracellular histones resulted in a significant increase in platelet adhesion levels in mouse cremaster venules compared to saline control. * p < 0.05. Treating vWF deficient mice (n = 9) with extracellular histones did not result in increased levels of platelet adhesion in cremaster venules. Each point represents an individual animal, with platelet adherence averaged over two separate venules per animal.

Histone-induced thrombocytopenia in vivo is independent of vWF and TLR4

Following visualization of platelet adherence in the mouse cremaster microvasculature, whole blood samples were collected from mice and assessed for platelet counts (Figure 3). Histone administration resulted in thrombocytopenia in all strains including wild-type mice, TLR4-deficient mice, vWF-deficient mice, and their littermate controls.

Figure 3. Intravascular histones result in thrombocytopenia in the presence or absence of vWF and TLR4.

Figure 3

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Following intravital microscopy of cremaster microvessels, blood was collected and analyzed for platelet concentrations using a veterinary hematology analyzer. In all cases, stimulation with extracellular histones resulted in thrombocytopenia defined as a significant decrease in blood platelet concentration. * p < 0.05. ** p < 0.01.

Extracellular histone-induced platelet aggregation is independent of platelet-derived vWF

As vWF is expressed by endothelial cells and platelets, we sought to determine whether the effect of histones on platelets required vWF. To pursue this, histone-induced platelet aggregation was measured on platelets isolated from vWF-deficient animals and their littermate controls. Collagen and saline were used as a positive and negative controls, respectively (Figure 4). In all mice, extracellular histones increased platelet aggregation, regardless of the presence of vWF. As compared to littermate control mice, vWF deficient mice showed no difference in final platelet aggregation in response to either histones (12.95 ± 0.18% vs 11.76 ± 0.62% aggregation), collagen (68.20 ± 1.36% vs 64.93 ± 3.35% aggregation), or saline control (0.23 ± 0.69% vs 0 ± 0.46% aggregation).

Figure 4. Histone stimulated platelet aggregation is independent of platelet derived vWF.

Figure 4

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Washed platelets were isolated from vWF deficient mice and their littermates (n = 4 per genotype) and stimulated with either extracellular histones (0.75 mg/ml), collagen (2 μg/ml) as a positive control, or saline as a negative control (A). Stimulation with extracellular histones resulted in a significant increase in platelet aggregation independent of platelet derived vWF (B). ** p < 0.01. *** p < 0.001. **** p < 0.0001.

Discussion

The primary finding in this study is that vWF is required for histone-induced platelet adhesion to microvessels in vivo. Furthermore, platelet-derived vWF is not necessary for histone-induced platelet aggregation ex vivo. Taken together, these data are consistent with the notion that endothelial vWF mediates histone-induced platelet activation in the microvasculature. While histones are normally intra-nuclear, they can be released into the extracellular space under cell death conditions in addition to NETosis [3–7]. Extracellular histones found in disease and injury are the lysine-rich H1, H2a and H2b, and the arginine-rich H3 and H4 [48]. This release of extracellular histones has been associated with inflammation and thrombosis and has been suggested as a predictor of poor outcome in sepsis [49,48,50]. It has been shown that extracellular histones result in vWF release from both platelet α-granules as well as endothelial Weibel-Palade bodies [51,10]. Histones induce endothelial cell release of Weibel-Palade bodies in a manner that is caspase-, calcium- and charge-dependent, thus promoting the capture of platelets by endothelium-bound ultra-long von Willebrand factor [13,10]. Histone exposure leads to platelet activation and subsequent vWF release; it has been theorized that the released vWF re-associates with platelets and provides additional binding sites for histones [25]. Of interest, our study shows that platelet-derived vWF is not necessary for histone-induced platelet aggregation ex vivo. Coupled with the fact that histones did not enhance platelet adhesion in vivo in vWF-deficient mice, this finding is consistent with the notion that endothelial-derived vWF mediates histone-induced platelet adhesion to microvessels in vivo.

In a previous study, our lab demonstrated the capacity of histones to bind to vascular endothelial cells in vitro, stimulating the release of ultra-large vWF and the formation of endothelial cell-bound ultra-large vWF strings[13]. The formation of endothelial cell-bound ultra-large vWF strings co-localized with histones is consistent with reports that histones are capable of binding to the A1 domain of vWF[41]. Moreover, these ultra-large vWF strings were capable of capturing lyophilized platelets (incapable of releasing vWF) in an in vitro flow chamber study, providing further evidence that endothelial-cell derived vWF plays an important role in histone-induced platelet-endothelial interaction. While platelets provide an alternate source of vWF, here we show that platelet-derived vWF is not necessary for histone-induced platelet aggregation despite increased levels of platelet adhesion to microvessels in mice treated with histones.

Histones have been reported to give rise to a procoagulant phenotype in human platelets, resulting in enhanced thrombin generation and rapid clot formation [18]. In addition, TLR4 has been shown to play a role in histone-induced endothelium activation in response to histones [52]. And while it has been shown that platelet TLR4 plays a role in histone-mediated platelet activation by inducing calcium-influx, recruitment of fibrin, and activation of pro-inflammatory signaling pathways [51], here we provide evidence that TLR4 is not necessary for histone-induced platelet adhesion to microvessels in vivo. Semeraro et al. previously demonstrated that histone-mediated platelet activation is, in part, dependent on both TLR2 and TLR4 [12]. However, their study did not study the role of TLR2 and TLR4 on platelet-endothelial interaction in vivo. While our findings demonstrate that TLR4 is not necessary for histone-induced platelet adhesion to microvessels in vivo, a limitation of our study is that we cannot exclude a role for TLR2 in these responses. Further studies will be necessary to define the role of TLR2 in histone-induced platelet activation and adhesion to microvessels in vivo.

In this study, vWF was not required for extracellular histone-induced thrombocytopenia. This is consistent with prior studies showing that histones induce rapid and profound thrombocytopenia in mice independent of vWF [25,10]. Histone administration in mice can result in endothelial injury, vascular barrier disruption and can contribute to multiple organ dysfunction and failure [5,21]. Furthermore, histone-induced platelet-endothelial interaction is not required for resultant tissue damage, though this interaction does play a large role in extracellular histone-induced death [3,25]. It is important to note that defining platelet adhesion in vivo is significantly more complex than in studies performed ex vivo. In vivo, platelets may adhere directly to endothelial cells, to each other, or to other blood cells; the measures of platelet adhesion to microvessels in vivo reported in our study do not allow distinction between these mechanisms. Additional studies are warranted to define the relative contribution of these adhesive interactions to the histone-induced platelet adhesion to microvessels in vivo.

Studies have demonstrated that histones can directly induce endothelial cell expression of tissue factor mediated by TLR2 and −4 activation. It is possible that this direct interaction between histones and the vascular endothelium in specific organs may play a role in the histone-induced thrombocytopenia, as histones have been demonstrated to cause lung and kidney damage associated with platelet sequestration in these respective tissues [26,10,12]. To this end, histones have been demonstrated to exacerbate lung injury in chronic obstructive pulmonary disease by stimulating lung cell apoptosis [53]. These interactions may lead to sequestration of platelets in organ tissues in a manner independent of histone-platelet association, which does not require vWF participation.

In summary, our study demonstrates that vWF is required for histone-induced platelet adhesion to microvessels in vivo. Histones induced similar degree of aggregation ex vivo in platelets derived from vWF-deficient mice and controls. While TLR4 has been shown to play a role in various histone-induced responses, under our experimental conditions, TLR4 was not required for histone-induced platelet adhesion to microvessels in vivo. The data presented in this paper are consistent with the notion that endothelium-derived vWF mediates histone-induced platelet adhesion in vivo. Additional studies are warranted to define the role of endothelial vWF release on platelet-microvessel interactions in conditions characterized by enhanced release of extracellular histones.

Perspectives

vWF, but not TLR4, was required for histone-induced platelet adhesion to mouse cremaster venules in vivo. Furthermore, platelet-derived vWF is not necessary for histone-induced platelet aggregation ex vivo. Taken together, these data are consistent with the notion that endothelial vWF mediates histone-induced platelet activation in the microvasculature.

Supplementary Material

Supplemental Figure 1

Supplemental Figure 1

Vascular Tracing of Mouse Cremaster Microvasculature and 3rd order venules. The vasculature of each animal was carefully traced during the equilibration period in order to accurately identify 3rd order venules for video capture. 1st, 2nd and 3rd order venules are identified (arrows), and examples of venules identified for video capture can be seen (circles).

Supplemental Video 1

Supplemental Video 1

Wild-type comparison of cremaster microvessels in mice treated with histones or saline. Representative videos of cremaster microvessels in wild-type mice treated with histones (top panel) or saline (bottom panel).

Download video file (5.6MB, mp4)
Supplemental Figure Legends

Acknowledgements

We would like to thank Ngoc-Anh Bui-Thanh for her excellent technical assistance with the cremaster microvascular thrombosis model. This work was supported in part by Merit Review Award I01 BX002551 from the Department of Veterans Affairs Biomedical Laboratory Research & Development Service (RER). JAC was supported by the NIH/NHLBI T32 training program HL139425. The content is solely the responsibility of the authors and does not represent the official views of the Department of Veterans Affairs or the United States government.

Sources of Support:

Department of Veterans Affairs Biomedical Laboratory Research & Development Service, Merit Review Award I01 BX002551 (RER) & NIH/NHLBI T32 training program grant HL139425 (JAC).

Abbreviations:

ACD

Acid Citrate Dextrose

CTH

Calf Thymus Histone

DAMP

Damage-Associated Molecular Pattern

DNA

Deoxyribonucleic Acid

EDTA

Ethylenediaminetetraacetic Acid

GP1bβ

Platelet Glycoprotein 1b Beta

IP

Intraperitoneal

NET

Neutrophil Extracellular Traps

PBS

Phosphate-Buffered Saline

PGI2

Prostacyclin

RCF

Relative Centrifugal Field

TLR

Toll-Like Receptor

vWF

von Willebrand Factor

WT

Wild-Type Mouse

Footnotes

Conflicts of Interest

The authors have no conflicts of interest.

Ethical Compliance: All applicable international, national, and institutional guidelines for the care and use of animals were followed.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, RER, upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure 1

Supplemental Figure 1

Vascular Tracing of Mouse Cremaster Microvasculature and 3rd order venules. The vasculature of each animal was carefully traced during the equilibration period in order to accurately identify 3rd order venules for video capture. 1st, 2nd and 3rd order venules are identified (arrows), and examples of venules identified for video capture can be seen (circles).

NIHMS1849040-supplement-Supplemental_Figure_1.tif (4MB, tif)
Supplemental Video 1

Supplemental Video 1

Wild-type comparison of cremaster microvessels in mice treated with histones or saline. Representative videos of cremaster microvessels in wild-type mice treated with histones (top panel) or saline (bottom panel).

Download video file (5.6MB, mp4)
Supplemental Figure Legends
NIHMS1849040-supplement-Supplemental_Figure_Legends.docx (12.5KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, RER, upon reasonable request.

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