Immunoreactivity and a new staining method of monocarboxylate transporter 1 located in endothelial cells of cerebral vessels of human brain in distinguishing cerebral venules from arterioles.

Distinguishing brain venules from arterioles with arteriolosclerosis is less reliable using traditional staining methods. We aimed to immunohistochemically assess the monocarboxylate transporter 1 (MCT1), a specific marker of venous endothelium found in rodent studies, in different caliber vessels in human brains. Both largeand small-caliber cerebral vessels were dissected from four autopsy donors. Immunoreactivity for MCT1 was examined in all autopsied human brain tissues, and then each vessel was identified by neuropathologists using hematoxylin and eosin stain, the Verhoeff's Van Gieson stain, immunohistochemical stain with antibodies for α-smooth muscle actin and MCT1 in sequence. A total of 61 cerebral vessels, including 29 arteries and 32 veins were assessed. Immunoreactivity for MCT1 was observed in the endothelial cells of various caliber veins as well as the capillaries, whereas that was immunenegative in the endothelium of arteries. The different labeling patterns for MCT1 could aid in distinguishing various caliber veins from arteries, whereas assessment using the vessel shape, the internal elastic lamina, and the pattern of smooth muscle fibers failed to make the distinction between small-caliber veins and sclerotic arterioles. In conclusion, MCT1 immunohistochemical staining is a sensitive and reliable method to distinguish cerebral veins from arteries.

The Conspicuousness of High Endothelial Venules in Angioimmunoblastic T-cell Lymphoma Is Due to Increased Cross-sectional Area, Not Increased Distribution Density.

Angioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma of follicular helper T-cell origin. Histologically, neoplastic T-cells proliferate to form clusters adjacent to or between arborizing high endothelial venules (HEVs). HEVs in normal lymph nodes express sulfated glycans called peripheral lymph node addressin (PNAd); however, it remains unclear whether PNAd is also expressed on HEVs in AITL. Furthermore, although it is widely accepted that HEVs are conspicuous in AITL due to their proliferation, quantitative histological support for this concept is lacking. To investigate these issues, we employed monoclonal antibodies recognizing PNAd, namely, MECA-79, HECA-452, and 297-11A, and performed quantitative immunohistochemical analysis of HEVs in 36 AITL-affected and 67 normal lymph nodes. Staining with all three antibodies confirmed that AITL HEVs express PNAd. Moreover, AITL HEVs were bound calcium-dependently by L-selectin-IgM fusion proteins, indicating that they function in the recruitment of L-selectin-expressing lymphocytes. Unexpectedly, HEV distribution density was not increased but rather decreased in AITL compared with normal lymph nodes, but HEV cross-sectional area in AITL was significantly greater than that seen in normal lymph nodes. Overall, these results indicate that the prominence of AITL HEVs is likely due to increased cross-sectional area rather than increased distribution density.

Single-cell transcriptome profiling reveals vascular endothelial cell heterogeneity in human skin.

Vascular endothelial cells (ECs) are increasingly recognized as active players in intercellular crosstalk more than passive linings of a conduit for nutrition delivery. Yet, their functional roles and heterogeneity in skin remain uncharacterized. We have used single-cell RNA sequencing (scRNA-seq) as a profiling strategy to investigate the tissue-specific features and intra-tissue heterogeneity in dermal ECs at single-cell level. Methods: Skin tissues collected from 10 donors were subjected to scRNA-seq. Human dermal EC atlas of over 23,000 single-cell transcriptomes was obtained and further analyzed. Arteriovenous markers discovered in scRNA-seq were validated in human skin samples via immunofluorescence. To illustrate tissue-specific characteristics of dermal ECs, ECs from other human tissues were extracted from previously reported data and compared with our transcriptomic data. Results: In comparison with ECs from other human tissues, dermal ECs possess unique characteristics in metabolism, cytokine signaling, chemotaxis, and cell adhesions. Within dermal ECs, 5 major subtypes were identified, which varied in molecular signatures and biological activities. Metabolic transcriptome analysis revealed a preference for oxidative phosphorylation in arteriole ECs when compared to capillary and venule ECs. Capillary ECs abundantly expressed HLA-II molecules, suggesting its immune-surveillance role. Post-capillary venule ECs, with high levels of adhesion molecules, were equipped with the capacity in immune cell arrest, adhesion, and infiltration. Conclusion: Our study provides a comprehensive characterization of EC features and heterogeneity in human dermis and sets the stage for future research in identifying disease-specific alterations of dermal ECs in various dermatoses.

Single-Cell Analysis Reveals Heterogeneity of High Endothelial Venules and Different Regulation of Genes Controlling Lymphocyte Entry to Lymph Nodes.

High-endothelial venules (HEVs) are specialized blood vessels allowing recirculation of naive lymphocytes through lymphoid organs. Here, using full-length, single-cell RNA sequencing, RNA fluorescence in situ hybridization (FISH), flow cytometry, and immunohistofluorescence, we reveal the heterogeneity of HEVs in adult mouse peripheral lymph nodes (PLNs) under conditions of homeostasis, antigenic stimulation, and after inhibition of lymphotoxin-ß receptor (LTßR) signaling. We demonstrate that HEV endothelial cells are in an activated state during homeostasis, and we identify the genes characteristic of the differentiated HEV phenotype. We show that LTßR signaling regulates many HEV genes and pathways in resting PLNs and that immune stimulation induces a global and temporary inflammatory phenotype in HEVs without compromising their ability to recruit naive lymphocytes. Most importantly, we uncover differences in the regulation of genes controlling lymphocyte trafficking, Glycam1, Fut7, Gcnt1, Chst4, B3gnt3, and Ccl21a, that have implications for HEV function and regulation in health and disease.

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.

Ca2+ Signalling in Pericytes.

Microcirculation is the generic name for the finest level of the circulatory system and consists of arteriolar and venular networks located upstream and downstream of capillaries, respectively. Anatomically arterioles are surrounded by a monolayer of spindle-shaped smooth muscle cells (myocytes), while terminal branches of precapillary arterioles, capillaries and all sections of postcapillary venules are surrounded by a monolayer of morphologically different perivascular cells (pericytes). Pericytes are essential components of the microvascular vessel wall. Wrapped around endothelial cells, they occupy a strategic position at the interface between the circulating blood and the interstitial space. There are physiological differences in the responses of pericytes and myocytes to vasoactive molecules, which suggest that these two types of vascular cells could have different functional roles in the regulation of local blood flow within the same microvascular bed. Also, pericytes may play different roles in different microcirculatory beds to meet the characteristics of individual organs. Contractile activity of pericytes and myocytes is controlled by changes of cytosolic free Ca2+concentration. In this chapter, we attempt to summarize the results in the field of Ca2+ signalling in pericytes especially in light of their contractile roles in different tissues and organs. We investigate the literature and describe our results regarding sources of Ca2+, relative importance and mechanisms of Ca2+ release and Ca2+ entry in control of the spatio-temporal characteristics of the Ca2+ signals in pericytes, where possible Ca2+ signalling and contractile responses in pericytes are compared to those of myocytes.

YAP Controls Endothelial Activation and Vascular Inflammation Through TRAF6.

RATIONALE: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. OBJECTIVE: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. METHODS AND RESULTS: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell-specific deletion of YAP. CONCLUSIONS: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.

Roles of Telocytes in the Development of Angiogenesis.

Telocytes are cells with telopodes, which distinguish them from other interstitial cells. According to the study of lung, it was confirmed that telocytes were mainly distributed in the alveolar interstitial tissues connected tightly with alveolar epithelia cells and participated in the structure of air-blood barrier, in the small vein and bronchioles and in the interstitial space of smooth muscle participated in the frame structure of the blood and bronchioles. Telocytes are positive to CD34 and C-kit which expressed on the surface of hemopoietic stem cells, and are proposed to participate in the angiogenesis. In this chapter, we try to clarify the morphological characteristics of lung telocytes, both in tissues and culture, and introduce the experiences on the method of telocytes isolation and primary culture. The proteomics analysis of lung telocytes through iTRAQ (isobaric tags for relative and absolute quantification) was also discussed and it will provide new research directions in the future.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1ß, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space.

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo.

UNLABELLED: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

Absence of Nkx2-3 homeodomain transcription factor reprograms the endothelial addressin preference for lymphocyte homing in Peyer's patches.

Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin ß receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.

Complete blood count and retinal vessel calibers.

OBJECTIVE: The influence of hematological indices such as complete blood count on microcirculation is poorly understood. Retinal microvasculature can be directly visualized and vessel calibers are associated with a range of ocular and systemic diseases. We examined the association of complete blood count with retinal vessel calibers. METHODS: Cross-sectional population-based Blue Mountains Eye Study, n = 3009, aged 49+ years. Complete blood count was measured from fasting blood samples taken at baseline examination, 1992-4. Retinal arteriolar and venular calibers were measured from digitized retinal photographs using a validated semi-automated computer program. RESULTS: All analyses adjusted for age, sex, systolic blood pressure, diabetes, smoking and fellow vessel caliber. Higher hematocrit, white cell count and platelet count were associated with narrower arteriolar caliber (p = 0.02, 0.03 and 0.001 respectively), while higher hemoglobin, hematocrit, red cell count, white cell count and platelet count were associated with wider venular caliber (p<0.0001 for all). Each quintile increase in hematocrit, white cell count and platelet count was associated with approximately 0.5 µm narrower arteriolar caliber; whereas each quintile increase in all of the complete blood count components was associated with approximately 1-2 µm wider venular caliber. CONCLUSIONS: These associations show that elevated levels of hematological indices can have adverse effects on the microcirculation.

A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment.

Neutrophil extravasation occurs across postcapillary venules, structures composed of endothelial cells (ECs), pericytes (PCs), and basement membrane (BM). We constructed composite models of the human postcapillary venule, combining ECs with PCs or PC-deposited BM, to better study this process. Quiescent and tumor necrosis factor α (TNF-α)-activated composites demonstrated in situ-like expression of cadherins, E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet-endothelial cell adhesion molecule 1 (PECAM-1), CD99, and interleukin 8 (IL-8). After TNF-α activation, the ECs supported greater neutrophil adhesion (66.1 vs. 23.7% of input cells) and transmigration (35.1 vs. 7.20% of input cells) than did the PCs, but the composites behaved comparably (no significant difference) to ECs in both assays. TNF-α-activated EC-conditioned medium (CM) increased transmigration across the PCs, whereas TNF-α-activated PC-CM decreased transmigration across the ECs, and culturing on PC-derived BM decreased both adhesion to and transmigration across the ECs. Anti-very late antigen 4 (VLA-4; on neutrophils) inhibited adhesion to TNF-α-activated composites, but not to ECs alone. Anti-CD99 (expressed on all 3 cell types) inhibited transmigration across the composites (14.5% of control) more than across the ECs (39.0% of control), and venular shear stress reduced transmigration across the ECs (17.3% of static) more than across the composites (36.7% of static). These results provide proof of concept that our composite human EC/PC/BM venular construct can reveal new interactions in the inflammatory cascade.

A prototype hyperspectral system with a tunable laser source for retinal vessel imaging.

PURPOSE: To describe the technology and determine the within-session repeatability of manual retinal reflectance measurements of arterioles and venules using a prototype hyperspectral retinal camera. METHODS: Six healthy young volunteers (three males, average age 26 ± 4 years) had five repeated sets of retinal images captured between 500 and 600 nm at 5-nm intervals using a newly developed hyperspectral retinal camera. Optical densities were manually extracted for first-degree arterioles and venules and the repeatability of retinal reflectance was compared sequentially. The SDs of the differences between sequential mean values were used as an indication of the variance, while the coefficient of repeatability (COR) and intraclass correlation coefficient (ICC) were used to assess repeatability. RESULTS: The mean difference between each sequential measure was calculated using 21 images from each of the five spectral cubes. The SDs of these values ranged from 0.01 to 0.06 OD units and from 0.01 to 0.07 OD units for first-degree arterioles and venules, respectively. The COR ranged from 0.02 to 0.11 OD units (relative to a mean OD of 0.15 [0.06-0.23] OD units) for arterioles and 0.03 to 0.14 OD units (relative to a mean OD of 0.25 [0.17-0.31] OD units) for venules. Good reliability (P < 0.001) was found for arterioles (ICC: 78.8%-94.4% with a Cronbach's α of 89.6%-97.6%) and for venules (ICC: 63.7%-92.1% with a Cronbach's α of 86.2%-98.1%). CONCLUSIONS: Manual optical density determination with this novel hyperspectral camera showed very good intrasession (and intraobserver) repeatability with a small degree of variance that should form the basis of reliable retinal oxygen saturation values in future imaging research studies. Future automation of retinal vessel reflectance image analyses will likely further improve this repeatability.

Perivascular hair follicle stem cells associate with a venule annulus.

The perivascular microenvironment helps in maintaining stem cells in many tissues. We sought to determine whether there is a perivascular niche for hair follicle stem cells. The association of vessels and follicle progenitor cells began by embryonic day 14.5, when nascent hair placodes had blood vessels approaching them. By birth, a vascular annulus stereotypically surrounded the keratin 15 negative (K15-) stem cells in the upper bulge and remained associated with the K15- upper bulge throughout the hair cycle. The angiogenic factor Egfl6 was expressed by the K15- bulge and was localized adjacent to the vascular annulus, which comprised post-capillary venules. Although denervation altered the phenotype of upper bulge stem cells, the vascular annulus persisted in surgically denervated mouse skin. The importance of the perivascular niche was further suggested by the fact that vascular annuli formed around the upper bulge of de novo-reconstituted hair follicles before their innervation. Together, these findings demonstrate that the upper bulge is associated with a perivascular niche during the establishment and maintenance of this specialized region of hair follicle stem cells.

The endothelial glycocalyx in syndecan-1 deficient mice.

The existence of a hydrodynamically relevant endothelial glycocalyx has been established in capillaries, venules, and arterioles in vivo. The glycocalyx is thought to consist primarily of membrane-bound proteoglycans with glycosaminoglycan side-chains, membrane-bound glypicans, and adsorbed plasma proteins. The proteoglycans found on the luminal surface of endothelial cells are syndecans-1, -2, and -4, and glypican-1. The extent to which any of these proteins might serve to anchor the glycocalyx to the endothelium has not yet been determined. To test whether syndecan-1, in particular, is an essential anchoring protein, we performed experiments to determine the hydrodynamically relevant glycocalyx thickness in syndecan-1 deficient (Sdc1(-/-)) mice. Micro-particle image velocimetry data were collected using a previously described method. Microviscometric analysis of these data consistently revealed the existence of a hydrodynamically relevant endothelial glycocalyx in Sdc1(-/-) mice in vivo. The mean glycocalyx thickness found in Sdc1(-/-) mice was 0.45±0.10 µm (N=15), as compared with 0.54±0.12 µm (N=11) in wild-type (WT) mice (p=0.03). The slightly thinner glycocalyx observed in Sdc1(-/-) mice relative to WT mice may be due to the absence of syndecan-1. These findings show that healthy Sdc1(-/-) mice are able to synthesize and maintain a hydrodynamically relevant glycocalyx, which indicates that syndecan-1 is not an essential anchoring protein for the glycocalyx in Sdc1(-/-) mice. This may also be the case for WT mice; however, Sdc1(-/-) mice might adapt to the lack of syndecan-1 by increasing the expression of other proteoglycans. In any case, syndecan-1 does not appear to be a prerequisite for the existence of an endothelial glycocalyx.

Shear-dependent adhesion of leukocytes and lectins to the endothelium and concurrent changes in thickness of the glycocalyx of post-capillary venules in the low-flow state.

OBJECTIVE: To elucidate shear-dependent effects of deformation of the endothelial glycocalyx on adhesion of circulating ligands in post-capillary venules, and delineate effect of MMPs. METHODS: Adhesion of WBCs and lectin-coated FLMs (0.1 µm diameter) to EC of post-capillary venules in mesentery was examined during acute reductions in shear rates (γ·, hemorrhagic hypotension). Adhesion was examined with or without superfusion with 0.5 µm doxycycline to inhibit MMPs. Thickness of the glycocalyx was measured by exclusion of fluorescent 70 kDa dextran from the EC surface. RESULTS: During superfusion with Ringers, rapid reductions in γ· resulted in a significant rise in WBC adhesion and a twofold rise in microsphere adhesion. With addition of doxycycline WBC and FLM adhesion increased twofold under high- and low-flow conditions. FLM adhesion was invariant with γ· throughout the network in the normal (high)-flow state. With reductions in γ·, thickness of the glycocalyx increased significantly, with or without doxycycline. CONCLUSIONS: The concurrent increase in WBC and FLM adhesion with increased thickness of the glycocalyx during reductions in shear suggests that glycocalyx core proteins recoil from their deformed steady-state configuration, which increases exposure of binding sites for circulating ligands.

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder.

BACKGROUND AND PURPOSE: In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca(2+) transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder. EXPERIMENTAL APPROACH: The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca(2+) dynamics were visualized using Fluo-4 fluorescence Ca(2+) imaging. KEY RESULTS: A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca(2+) transients, which were accompanied by phasic venular constrictions. Nicardipine (1 µM) disrupted the synchrony of spontaneous Ca(2+) transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca(2+) transients were suppressed by cyclopiazonic acid (10 µM), 2-aminoethoxydiphenyl borate (10 µM), U-73122 (1 µM), oligomycin (1 µM) and SKF96365 (10 µM), but unaffected by ryanodine (100 µM) or YM-244769 (1 µM), suggesting that pericyte Ca(2+) transients rely on Ca(2+) release from the endoplasmic reticulum via the InsP(3) receptor and also require Ca(2+) influx through store-operated Ca(2+) channels. CONCLUSIONS AND IMPLICATIONS: The pericytes in mouse bladder can generate spontaneous Ca(2+) transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.

S-Nitrosation of ß-catenin and p120 catenin: a novel regulatory mechanism in endothelial hyperpermeability.

RATIONALE: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. OBJECTIVE: We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. METHODS AND RESULTS: We measured PAF-stimulated SNO of ß-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of ß-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-α increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of ß-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin. CONCLUSIONS: Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.

Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat.

The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression.