Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection.

Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and 'instruct' them with pattern-recognition and motility programs.

Coordinated navigation within tissues is essential for cells of the innate immune system to reach the sites of inflammatory processes, but the signals involved are incompletely understood. Here we demonstrate that NG2(+) pericytes controlled the pattern and efficacy of the interstitial migration of leukocytes in vivo. In response to inflammatory mediators, pericytes upregulated expression of the adhesion molecule ICAM-1 and released the chemoattractant MIF. Arteriolar and capillary pericytes attracted and interacted with myeloid leukocytes after extravasating from postcapillary venules, 'instructing' them with pattern-recognition and motility programs. Inhibition of MIF neutralized the migratory cues provided to myeloid leukocytes by NG2(+) pericytes. Hence, our results identify a previously unknown role for NG2(+) pericytes as an active component of innate immune responses, which supports the immunosurveillance and effector function of extravasated neutrophils and macrophages.

High endothelial venules as potential gateways for therapeutics.

High endothelial venules (HEVs) are specialized blood vessels that support the migration of lymphocytes from the bloodstream into lymph nodes (LNs). They are also formed ectopically in mammalian organs affected by chronic inflammation and cancer. The recent arrival of immunotherapy at the forefront of many cancer treatment regimens could boost a crucial role for HEVs as gateways for the treatment of cancer. In this review, we describe the microanatomical and biochemical characteristics of HEVs, mechanisms of formation of newly made HEVs, immunotherapies potentially dependent on HEV-mediated T cell homing to tumors, and finally, how HEV-targeted therapies might be used as a complementary approach to potentially shape the therapeutic landscape for the treatment of cancer and immune-mediated diseases.

Obliteration of portal venules contributes to portal hypertension in biliary cirrhosis.

The effects of the obliteration of portal venules (OPV) in cirrhotic portal hypertension are poorly understood. To investigate its contribution to portal hypertension in biliary cirrhosis and its underlying mechanism, we evaluated OPV using two-dimensional (2D) histopathology in liver explants from patients with biliary atresia (BA, n = 63), primary biliary cholangitis (PBC, n = 18), and hepatitis B-related cirrhosis (Hep-B-cirrhosis, n = 35). Then, three-dimensional (3D) OPV was measured by X-ray phase-contrast CT in two parallel models in rats following bile duct ligation (BDL) or carbon tetrachloride (CCl4) administration, representing biliary cirrhosis and post-necrotic cirrhosis, respectively. The portal pressure was also measured in the two models. Finally, the effects of proliferative bile ducts on OPV were investigated. We found that OPV was significantly more frequent in patients with biliary cirrhosis, including BA (78.57 ± 16.45%) and PBC (60.00 ± 17.15%), than that in Hep-B-cirrhotic patients (29.43 ± 14.94%, p < 0.001). OPV occurred earlier, evidenced by the paired liver biopsy at a Kasai procedure (KP), and was irreversible even after a successful KP in the patients with BA. OPV was also significantly more frequent in the BDL models than in the CCl4 models, as shown by 2D and 3D quantitative analysis. Portal pressure was significantly higher in the BDL model than that in the CCl4 model. With the proliferation of bile ducts, portal venules were compressed and irreversibly occluded, contributing to the earlier and higher portal pressure in biliary cirrhosis. OPV, as a pre-sinusoidal component, plays a key role in the pathogenesis of portal hypertension in biliary cirrhosis. The proliferated bile ducts and ductules gradually take up the 'territory' originally attributed to portal venules and compress the portal venules, which may lead to OPV in biliary cirrhosis. © 2024 The Pathological Society of Great Britain and Ireland.

Human blood vessel organoids as a model of diabetic vasculopathy.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

PNAd-expressing vessels characterize the dermis of CD3+ T-cell-mediated cutaneous diseases.

T-cell recruitment to skin tissues is essential for inflammation in different cutaneous diseases; however, the mechanisms by which these T cells access the skin remain unclear. High endothelial venules expressing peripheral node address in (PNAd), an L-selectin ligand, are located in secondary lymphoid organs and are responsible for increasing T-cell influx into the lymphoid tissues. They are also found in non-lymphoid tissues during inflammation. However, their presence in different common inflammatory cutaneous diseases and their correlation with T-cell infiltration remain unclear. Herein, we explored the mechanisms underlying the access of T cells to the skin by investigating the presence of PNAd-expressing vessels in different cutaneous diseases, and its correlation with T cells' presence. Skin sections of 43 patients with different diseases were subjected to immunohistochemical and immunofluorescence staining to examine the presence of PNAd-expressing vessels in the dermis. The correlation of the percentage of these vessels in the dermis of these patients with the severity/grade of CD3+ T-cell infiltration was assessed. PNAd-expressing vessels were commonly found in the skin of patients with different inflammatory diseases. A high percentage of these vessels in the dermis was associated with increased severity of CD3+ T-cell infiltration (P < 0.05). Additionally, CD3+ T cells were found both around the PNAd-expressing vessels and within the vessel lumen. PNAd-expressing vessels in cutaneous inflammatory diseases, characterized by CD3+ T-cell infiltration, could be a crucial entry point for T cells into the skin. Thus, selective targeting of these vessels could be beneficial in cutaneous inflammatory disease treatment.

Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic.

Food intake in South African children and retinal microvascular health: The ExAMIN Youth SA study.

BACKGROUND AND AIMS: Retinal arteriolar narrowing and venular widening are associated with increased cardiovascular risk, even at young ages. Whether diet contributes to early microvascular changes in children is not widely explored. We explored the associations of frequency of healthy and unhealthy food group intake with retinal vessel calibers in black and white children. METHODS AND RESULTS: This study included school-aged (5-9 years) black (N = 433, 7.46 ± 0.98 years), and white (N = 403, 7.43 ± 0.82 years) children. Anthropometric and blood pressure measurements were taken, along with retinal vessel calibers (central retinal arteriolar (CRAE) and venular (CRVE) equivalents). Frequencies of food group intake were assessed using a food-frequency questionnaire. A factor analysis was performed to describe food group patters. Independent associations between retinal vessel calibers and frequencies of food group intake and food group patters were explored. In black children, cookies, cakes, and biscuits were associated with narrower arterioles (p < 0.05). In white children, cold sweetened beverages were associated with narrower arterioles (p = 0.02), whereas salty snacks were associated with narrower arterioles (p = 0.01) and wider venules (p < 0.05). Fruits were positively associated with CRAE (p = 0.03) in white children only. CONCLUSION: A higher frequency of unhealthy food group consumption was associated with retinal arteriolar narrowing and venular widening in both black and white children. However, fruit intake was shown beneficial for retinal microvascular health in white children only. Our findings may highlight the importance of promoting healthy eating patterns from early childhood which may reduce the risk of premature cardiovascular disease development.

RETINAL MICROVASCULAR CHANGES AND RISK OF CORONARY HEART DISEASE: A Systematic Review and Meta-Analysis.

PURPOSE: To quantify associations between various retinal microvascular changes and the risk of the development of coronary heart disease (CHD). METHODS: PubMed, Embase, Web of Science, and Cochrane Library databases were searched for cohort studies on the association between retinal microvascular changes and incident CHD up to July 31, 2023. The summary risk estimates were estimated using the random-effects model. Subgroup and sensitivity analyses were performed to investigate the potential source of heterogeneity. RESULTS: The authors identified 21 studies that met the inclusion criteria of this meta-analysis through database searching. This study yielded significant associations between retinal microvascular changes, including arteriolar narrowing, venular widening, vessel occlusion, and other retinal vascular signs, and the risk of CHD, with pooled adjusted hazard ratios of 1.20 (95% confidence interval: 1.13-1.27). In sex- and age-stratified analyses, retinal microvascular changes were associated with a greater risk of developing CHD in female patients and younger adults. CONCLUSION: A range of retinal microvascular changes was associated with the risk of CHD, particularly in female patients and younger ages. The results of this study support the concept that retinal microvascular abnormalities may be markers for future CHD. Noninvasive retinal microvascular assessments may be helpful in screening patients with increased CHD risk.

Control of low flow regions in the cortical vasculature determines optimal arterio-venous ratios.

The energy demands of neurons are met by a constant supply of glucose and oxygen via the cerebral vasculature. The cerebral cortex is perfused by dense, parallel arterioles and venules, consistently in imbalanced ratios. Whether and how arteriole-venule arrangement and ratio affect the efficiency of energy delivery to the cortex has remained an unanswered question. Here, we show by mathematical modeling and analysis of the mapped mouse sensory cortex that the perfusive efficiency of the network is predicted to be limited by low-flow regions produced between pairs of arterioles or pairs of venules. Increasing either arteriole or venule density decreases the size of these low-flow regions, but increases their number, setting an optimal ratio between arterioles and venules that matches closely that observed across mammalian cortical vasculature. Low-flow regions are reshaped in complex ways by changes in vascular conductance, creating geometric challenges for matching cortical perfusion with neuronal activity.

Density of high endothelial venules and PDL-1 expression: relationship with tumor-infiltrating lymphocytes in primary cutaneous melanomas.

Studies have highlighted melanoma immunogenicity, and the prognostic importance of tumor infiltrating lymphocytes (TILs) and mechanisms of tumor immune evasion, such as hyperexpression of programmed cell death ligand 1 (PDL-1). High endothelial venules (HEV) are specialized blood vessels that can facilitate the lymphocytes migration to the tumor. Here we evaluate the association of HEV density and PDL-1 expression in primary cutaneous melanomas with the presence and degree of TILs and with other clinicopathological variables (age, sex, tumor location, melanoma histological type, Breslow thickness, ulceration, regression signs, mitotic index). HEV density and PDL-1 expression were assessed immunohistochemically in 78 melanoma cases, using a specific antibody, and were detected in 59% and 76% of these, respectively. Positive associations were identified between HEV density and PDL-1 expression with the presence and degree of lymphocytic infiltration, melanoma histological type and ulceration presence. No correlation was found between HEV density and PDL-1 expression. Our findings confirm the HEV role in the recruitment and facilitation of lymphocyte transport in cutaneous melanomas, where HEV density is strongly associated with the degree of TILs. Additionally, PDL-1 hyperexpression suggests a possible mechanism of tumor immune evasion, which may lead to inactivation and reduction of the tumor lymphocytes number.

Modeling angiogenesis in the human brain in a tissue-engineered post-capillary venule.

Angiogenesis plays an essential role in embryonic development, organ remodeling, wound healing, and is also associated with many human diseases. The process of angiogenesis in the brain during development is well characterized in animal models, but little is known about the process in the mature brain. Here, we use a tissue-engineered post-capillary venule (PCV) model incorporating stem cell derived induced brain microvascular endothelial-like cells (iBMECs) and pericyte-like cells (iPCs) to visualize the dynamics of angiogenesis. We compare angiogenesis under two conditions: in response to perfusion of growth factors and in the presence of an external concentration gradient. We show that both iBMECs and iPCs can serve as tip cells leading angiogenic sprouts. More importantly, the growth rate for iPC-led sprouts is about twofold higher than for iBMEC-led sprouts. Under a concentration gradient, angiogenic sprouts show a small directional bias toward the high growth factor concentration. Overall, pericytes exhibited a broad range of behavior, including maintaining quiescence, co-migrating with endothelial cells in sprouts, or leading sprout growth as tip cells.

DC101, an anti-VEGFR2 agent, promotes high-endothelial venule formation and immune infiltration versus SAR131675 and fruquintinib.

There is an increasing interest in combining immune checkpoint inhibitors (ICIs) with anti-angiogenic drugs to enhance their anti-tumor effects. In this study, three anti-angiogenic agents, DC101 (acting on VEGFR2), SAR131675 (acting on VEGFR3), and fruquintinib (a small-molecule inhibitor acting on multiple targets) were administered to B16F1-OVA-loaded C57BL/6 mice. Immune cells infiltration in the tumor tissues, vascular normalization, and high-endothelial venule (HEV) formation were assessed to provide evidence for drug combination. Both DC101 and fruquintinib significantly slowed the melanoma growth and increased the proportion of CD3+ and CD8+ T cells infiltration compared with SAR131675, of note, the effect of DC101 was more pronounced. Moreover, DC101 and fruquintinib increased the interferon-γ and perforin levels, meanwhile, DC101 increased the granzyme B levels, whereas fruquintinib and SAR131675 did not. Only the fruquintinib-treated group showed decreased regulatory T cells infiltration. We found upregulation of PD-L1 expression in tumor cells and CD45+ immune cells in DC101-treated group as well as upregulation of PD-1 expression on CD3+ T cells. However, fruquintinib only increased PD-L1 expression in tumors. Both DC101 and fruquintinib reduced the proportion of CD31+ vessels, while DC101 increased the ratio of α-SMA +/CD31+ cells and reduced the expression of HIF-1α more than fruquintinib. Moreover, DC101 enhanced the infiltration of dendritic cells and B cells, and local HEV formation. In conclusion, our data indicate that DC101 may be a better choice for the combined clinical application of ICIs and anti-angiogenic agents.

Automated detection of vascular remodeling in tumor-draining lymph nodes by the deep-learning tool HEV-finder.

Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Retinal Venule Coverage by Pericytes Decreases in Multiparous Mice in a Time-Dependent Manner Post-Delivery.

Structural changes in the retinal vasculature have been linked to increased cardiovascular risks and also change as a function of age. Because multiparity has been associated with poorer cardiovascular health scores, we hypothesized that changes in retinal vascular caliber would be observed in multiparous, compared to nulliparous, females and retired breeder males. Age-matched nulliparous (n = 6) and multiparous (n = 11, retired breeder females with 4 ± 1 litters), and male breeder (n = 7) SMA-GFP reporter mice were included for assessment of retinal vascular structure. Multiparous females had higher body mass, heart weight, and kidney weight compared to nulliparous mice, with lower kidney and higher brain weight compared to male breeders. There was no difference in number of retinal arterioles or venules, or arteriole or venule diameter among groups; however, venous pericyte density (number per venule area) decreased in multiparous vs. nulliparous mice and was negatively associated with the time since last litter and with age. Our results suggest that the time elapsed since delivery is an important factor to be considered in multiparity studies. Taken together, changes in vascular structure and potentially function, are time- and age-dependent. Ongoing and future work will determine whether structural changes are associated with functional consequences at the blood-retinal barrier.

QUANTITATIVE MORPHOLOGICAL ASPECTS OF THE STUDY OF THE FEATURES OF THE REMODELING OF THE HEMOMICROCIRCULATORY CHANNEL OF THE TESTICLES UNDER THE ACTION OF ALUMINUM CHLORIDE ON THE BODY.

OBJECTIVE: The aim: To conduct a morphometric analysis of the features of the remodeling of vessels of the hemomicrocirculatory channel of the testicles under the action of aluminum chloride on the body. PATIENTS AND METHODS: Materials and methods: The testicles of 52 white male Wistar rats were morphologically studied, which were divided into two groups: intact and animals injected with aluminum chloride at a dose of 100 mg/kg. RESULTS: Results: The long-term effect of aluminum chloride on the body led to pronounced changes in the quantitative morphological indicators of the vessels of the hemomicrocirculatory channel of the testes compared to the control ones. The diameter of arterioles of the left testicle decreased by 18.4%, precapillary arterioles by 19.1%, hemocapillaries by 10.1%. The diameter of the postcapillary venules of the left testicle increased by 26.3%, the venules by 26.4%, and the density of microvessels decreased by 30.0%, which indicated the deterioration of its blood supply. The degree of remodeling of microvessels in the right testis was less pronounced. The diameter of arterioles of the right testicle decreased by 17.1%, precapillary arterioles by 18.4 %, and hemocapillaris by 9.1%. The diameter of the postcapillary venules of the right testicle increased by 25.5%, the venules by 27.0%, and the density of microvessels decreased by 25.8%. CONCLUSION: Conclusions: Long-term action (during the month) of aluminum chloride on the body leads to pronounced remodeling of the vessels of the hemomicrocirculatory channel of the testicles, which is characterized by a pronounced narrowing of its supply (arterioles, precapillary arterioles), exchange (hemocapillaries) links.

Perspective: The Case for Acute Large Vessel Ischemic Stroke in COVID-19 Originating Within Thrombosed Pulmonary Venules.

The main burden of SARS-CoV-2 falls on the lungs but neurological manifestations, the most disabling of which are strokes and which correlate with disease severity, are common. We proffer a novel mechanism for acute COVID-19 stroke whereby pulmonary vein clots developing within the characteristic pulmonary intravascular thrombotic lesions can embolize to the brain. Appreciation of this mechanism requires an understanding of the tricompartmental model of lung parenchyma oxygenation (the alveolus, the bronchial artery, and the pulmonary artery), all of which are compromised in COVID-19. Of these 3 sources, the bronchial artery plays a crucial role in COVID-19 stroke because the unique collaterals from bronchial artery to pulmonary vein which exist under normal physiological conditions (and which maintain venous patency when the pulmonary artery is blocked by embolus) are occluded, thus leading to venular thrombosis in the presence of hypercoagulability. Dislodgement of clots from this source translocates the pathology to the brain and is a disease mechanism, formerly rare, which may account for many cases of large vessel occlusion stroke in COVID-19. This mechanism extends the concept of cardioembolic stroke from endocardium retrogradely into the pulmonary circulation with which the left cardiac chambers lie in direct continuity, and which is an accepted stroke mechanism under other circumstances such as lung lobectomy, where surgical ligation of the pulmonary vein creates a blind sac from which thrombi can embolize. The proposed model is supported by postmortem studies which have demonstrated venular thrombosis and by case reports of pulmonary vein thrombosis in COVID-19. This concept provides a more plausible cause for COVID-19 associated large vessel occlusion stroke than other putative mechanisms, such as cerebral endotheliitis, cytokine storm, and hypercoagulopathy, although it is acknowledged that the latter mechanism contributes to the genesis of pulmonary vein clots. Recognizing that extrapulmonary manifestations including stroke arise within thrombosed pulmonary veins is key to understanding of neurological manifestations of SARS-CoV-2 infection.

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

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 pro-inflammatory 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.