An Intact Kidney Slice Model to Investigate Vasa Recta Properties and Function in situ

Background: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. Results: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow

Strong expression of TGF-beta in human host tissues around subcutaneous Dirofilaria repens

Dirofilaria repens and other Dirofilaria species are widely distributed parasitic nematodes of carnivores, which occasionally are transmitted to men, causing subcutaneous nodules. In humans, it usually occurs only as single male or female filariae without production of microfilariae. The non-productive living or dead Dirofilaria worms in subcutaneous biopsies from 15 human patients permitted us to study the role of the pleiotropic and immunoregulatory cytokine transforming growth factor beta (TGF-beta) independent from the influence of microfilariae. Antiserum against latent TGF-beta 1 was used for an immunohistological examination. In the infiltrates around female and male filariae, there occurred strongly TGF-beta-positive macrophages, mast cells, endothelial cells, fibrocytes, and giant cells adjacent to dead worms. In one nodule, secondary lymph follicles were observed with clearly TGF-beta-positive B cells in the mantle zone and weakly positive macrophages and B cells in the germinal centre. A network of CD35-positive follicular dendritic cells was observed in the germinal centre. All Dirofilaria contained Wolbachia endobacteria, which probably had attracted the numerous TGF-beta-negative neutrophils near to the worm. Wolbachia were phagocytosed by neutrophils adjacent to dead filariae. Macrophages and lymphocytes expressed the MHC class II molecule HLA-DR in small accumulations of immune cells in the outer zone of the infiltrate and the mantle zone and germinal centre of secondary lymph follicles. It is concluded that single non-productive Dirofilaria worms elicit a strong expression of TGF-beta. This result is in accordance with observations on Onchocerca volvulus from patients with the hyporeactive (generalised) form

A molecular atlas of cell types and zonation in the brain vasculature

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.Peer reviewe

p75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75NTR expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75NTR activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs. NF-κB further induces activation of Rho kinase and shedding of endothelial microparticles carrying miR-503, which transfer miR-503 from ECs to vascular pericytes. The integrin-mediated uptake of miR-503 in the recipient pericytes reduces expression of EFNB2 and VEGFA, resulting in impaired migration and proliferation. We confirm operation of the above mechanisms in mouse models of diabetes, in which EC-derived miR-503 reduces pericyte coverage of capillaries, increased permeability and impaired post-ischaemic angiogenesis in limb muscles. Collectively, our data demonstrate that miR-503 regulates pericyte–endothelial crosstalk in microvascular diabetic complications

VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFAinduced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms

Effects of PI and PIII Snake Venom Haemorrhagic Metalloproteinases on the Microvasculature: A Confocal Microscopy Study on the Mouse Cremaster Muscle

The precise mechanisms by which Snake Venom Metalloproteinases (SVMPs) disrupt the microvasculature and cause haemorrhage have not been completely elucidated, and novel in vivo models are needed. In the present study, we compared the effects induced by BaP1, a PI SVMP isolated from Bothrops asper venom, and CsH1, a PIII SVMP from Crotalus simus venom, on cremaster muscle microvasculature by topical application of the toxins on isolated tissue (i.e., ex vivo model), and by intra-scrotal administration of the toxins (i.e., in vivo model). The whole tissue was fixed and immunostained to visualize the three components of blood vessels by confocal microscopy. In the ex vivo model, BaP1 was able to degrade type IV collagen and laminin from the BM of microvessels. Moreover, both SVMPs degraded type IV collagen from the BM in capillaries to a higher extent than in PCV and arterioles. CsH1 had a stronger effect on type IV collagen than BaP1. In the in vivo model, the effect of BaP1 on type IV collagen was widespread to the BM of arterioles and PCV. On the other hand, BaP1 was able to disrupt the endothelial barrier in PCV and to increase vascular permeability. Moreover, this toxin increased the size of gaps between pericytes in PCV and created new gaps between smooth muscle cells in arterioles in ex vivo conditions. These effects were not observed in the case of CsH1. In conclusion, our findings demonstrate that both SVMPs degrade type IV collagen from the BM in capillaries in vivo. Moreover, while the action of CsH1 is more directed to the BM of microvessels, the effects of BaP1 are widespread to other microvascular components. This study provides new insights in the mechanism of haemorrhage and other pathological effects induced by these toxins

Glutaredoxin-1 Overexpression Enhances Neovascularization and Diminishes Ventricular Remodeling in Chronic Myocardial Infarction

Oxidative stress plays a critical role in the pathophysiology of cardiac failure, including the modulation of neovascularization following myocardial infarction (MI). Redox molecules thioredoxin (Trx) and glutaredoxin (Grx) superfamilies actively maintain intracellular thiol-redox homeostasis by scavenging reactive oxygen species. Among these two superfamilies, the pro-angiogenic function of Trx-1 has been reported in chronic MI model whereas similar role of Grx-1 remains uncertain. The present study attempts to establish the role of Grx-1 in neovascularization and ventricular remodeling following MI. Wild-type (WT) and Grx-1 transgenic (Grx-1Tg/+) mice were randomized into wild-type sham (WTS), Grx-1Tg/+ Sham (Grx-1Tg/+S), WTMI, Grx-1Tg/+MI. MI was induced by permanent occlusion of the LAD coronary artery. Sham groups underwent identical time-matched surgical procedures without LAD ligation. Significant increase in arteriolar density was observed 7 days (d) after surgical intervention in the Grx-1Tg/+MI group as compared to the WTMI animals. Further, improvement in myocardial functional parameters 30 d after MI was observed including decreased LVIDs, LVIDd, increased ejection fraction and, fractional shortening was also observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Moreover, attenuation of oxidative stress and apoptotic cardiomyocytes was observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Increased expression of p-Akt, VEGF, Ang-1, Bcl-2, survivin and DNA binding activity of NF-κB were observed in the Grx-1Tg/+MI group when compared to WTMI animals as revealed by Western blot analysis and Gel-shift analysis, respectively. These results are the first to demonstrate that Grx-1 induces angiogenesis and diminishes ventricular remodeling apparently through neovascularization mediated by Akt, VEGF, Ang-1 and NF-κB as well as Bcl-2 and survivin-mediated anti-apoptotic pathway in the infarcted myocardium

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption

Low-Dosage Inhibition of DII4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain