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Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. Objective: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype. Methods and Results: We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfb ret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfb ret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. Conclusions: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.

Several studies suggest that hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections. We hypothesized that the high levels of the inflammatory cytokine Angiopoietin-2 (ANGPT2) reported in hospitalized COVID-19 patients might promote hypercoagulation through ANGPT2 binding to thrombomodulin with resulting inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Plasma was collected from critically ill COVID-19 patients treated in the intensive care unit (ICU) at Uppsala University Hospital and ANGPT2 was measured at admission (61 patients) and after ten days (40 patients). ANGPT2 levels were compared with biochemical parameters, clinical outcome, and survival. We found that ANGPT2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. To test causality, we administered ANGPT2 to wildtype mice and found that it shortened bleeding time in a tail injury model. In further support of a role for ANGPT2 in physiological coagulation, bleeding time was increased in endothelial-specific Angpt2 knockout mice. Using in vitro assays, we found that ANGPT2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data reveal a novel mechanism for ANGPT2 in hypercoagulation and suggest that Angiopoietin-2 inhibition may be tested in the treatment of hypercoagulation in severe COVID-19 infection.

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies., The molecular identity of renal glomerular cells is poorly characterized and rodent glomerulopathy models translate poorly to humans. Here, the authors show molecular signatures of glomerulus-associated cells using single cell RNA sequencing and highlight differences between mouse and human cells.

The glomerulus is a highly specialized microvascular bed that filters blood to form primary urinary filtrate. It contains four cell types: fenestrated endothelial cells, specialized vascular support cells termed podocytes, perivascular mesangial cells, and parietal epithelial cells. Glomerular cell-cell communication is critical for the development and maintenance of the glomerular filtration barrier. VEGF, ANGPT, EGF, SEMA3A, TGF-β, and CXCL12 signal in paracrine fashions between the podocytes, endothelium, and mesangium associated with the glomerular capillary bed to maintain filtration barrier function. In this review, we summarize the current understanding of these signaling pathways in the development and maintenance of the glomerulus and the progression of disease.

Background Docetaxel is a cytostatic agent approved for treatment of non-small cell lung cancer as well as other cancers. Although docetaxel is an effective cytostatic agent, its effectiveness in clinical practice is associated with a variety of acute and long term side-effects. To overcome systemic side-effects, a slow release formulation based on calcium sulfate with docetaxel for intra-tumoral administration was developed. Methods Two formulations with the calcium sulfate NanoZolid technology were generated with a twofold difference in docetaxel drug load. The formulations were injected intra-tumorally as a paste which solidified within the tumor. The effects of the two intra-tumoral injection formulations were tested in female mice (n = 60) inoculated with subcutaneous Lewis lung carcinoma cells. The two formulations were compared to systemic intraperitoneal injection of docetaxel and a placebo formulation without docetaxel. Tumor volumes were measured and systemic side-effects were evaluated using body weight and cell counts from whole blood as well as plasma concentrations. Results Both docetaxel formulations showed a significantly higher antitumor efficacy compared to placebo, which was comparable to that of systemic administration of docetaxel. Moreover, the intra-tumoral formulations with docetaxel showed reduced systemic toxicity compared to systemic treatment, including less weight loss and no decrease in blood cell counts. Conclusions The results suggest that intra-tumoral slow release calcium sulfate based formulations with docetaxel can be an alternative strategy as an efficient local antitumoral treatment with reduced systemic toxicity.

Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angpt1 and Angpt2 (A1A2Flox(WB) mice) lacked drainage pathways in the corneal limbus, including Schlemm's canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2Flox(WB) mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium.

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a “tet-on” inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin–Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.

Aims/hypothesis Despite the fact that diabetic nephropathy is an increasingly common disorder that may lead to uraemia, the underlying mechanisms are still poorly understood and there is no specific therapy. To clarify whether long-term diabetes alters glomerular size- or charge-selectivity or both, we studied non-obese diabetic mice for up to 40 weeks. Materials and methods During the study period, spot urine was collected and blood pressure measured. At weeks 10 and 40, the right kidney was isolated and perfused at 8°C to inhibit tubular function, allowing for analysis of glomerular selectivity with albumin and Ficoll clearance. The left kidney was removed for further investigation using electron microscopy and molecular biology. Real-time PCR with low-density arrays was done to evaluate renal cortex mRNA expression of proteoglycans and other components in the glomerular barrier. After 40 weeks of diabetes, kidneys showed morphological changes typical of diabetic complications. Results At 40 weeks, the fractional clearance for negatively charged albumin was three times higher in the diabetic animals (0.0160) than in controls (0.0051, p

In this study, we pursued the somewhat controversial issue whether the glycosaminoglycans (GAG) in the endothelial cell glycocalyx are important for glomerular size and charge selectivity. In isoflurane-anesthetized mice, Intralipid droplets were used as indirect markers of the glomerular endothelial cell-surface layer, i.e., the glycocalyx. The mice were given intravenous injections of GAG-degrading enzymes, which due to their high molecular weight remained and acted intravascularly. Flow-arrested kidneys were fixed and prepared for electron microscopy, and the distance between glomerular endothelial cells and the luminal Intralipid droplets was measured. The relative frequency of Intralipid droplets was calculated for each 50-nm increment zone up to 500 nm from the endothelial cell membrane surface as were the mean distances. Glomerular size and charge selectivity were estimated from the clearance data for neutral Ficolls (molecular radii of 12-72 A), and albumin in isolated kidneys was perfused at 8 degrees C. In enzyme-treated animals (hyaluronidase, heparinase, and chondroitinase), the relative Intralipid droplet frequency in the zone closest to the endothelial cells, i.e., 0-50 nm, was increased approximately 2.5 times compared with controls. Also, the mean distance between the Intralipid droplets and the endothelium was decreased from 176 to 115-122 nm by enzyme treatment. These changes were accompanied by an increase in the fractional clearance for albumin. In conclusion, both morphological and functional measurements suggest the endothelial cell glycocalyx to be an important component of the glomerular barrier.

Vascular endothelial growth factor A (VEGFA) expression is increased in glomeruli in the context of diabetes. Here, we tested the hypothesis that this upregulation of VEGFA protects the glomerular microvasculature in diabetes and that therefore inhibition of VEGFA will accelerate nephropathy. To determine the role of glomerular Vegfa in the development and progression of diabetic nephropathy, we used an inducible Cre-loxP gene-targeting system that enabled genetic deletion of Vegfa selectively from glomerular podocytes of wild-type or diabetic mice. Type 1 diabetes was induced in mice using streptozotocin (STZ). We then assessed the extent of glomerular dysfunction by measuring proteinuria, glomerular pathology, and glomerular cell apoptosis. Vegfa expression increased in podocytes in the STZ model of diabetes. After 7 weeks of diabetes, diabetic mice lacking Vegfa in podocytes exhibited significantly greater proteinuria with profound glomerular scarring and increased apoptosis compared with control mice with diabetes or Vegfa deletion without diabetes. Reduced local production of glomerular Vegfa in a mouse model of type 1 diabetes promotes endothelial injury accelerating the progression of glomerular injury. These results suggest that upregulation of VEGFA in diabetic kidneys protects the microvasculature from injury and that reduction of VEGFA in diabetes may be harmful.

Deletion of the von Hippel-Lindau tumor suppressor ( Vhl) gene from renal podocytes of mice (pod Vhl KO) leads to rapidly progressive glomerulonephritis (RPGN), a clinical syndrome characterized by rapid loss of renal function and crescents on renal biopsy. Genomic profiling of glomeruli isolated from pod Vhl knockout (KO) mice and from patients with RPGN identified a fingerprint of genes regulated by hypoxia-inducible factors (HIF), important substrates of the product of the VHL gene. Here, we show that stabilization of Hifs in podocytes is both required and sufficient for the glomerular phenotype observed in pod Vhl KO mice. Genetic deletion of the obligate dimerization partner Arnt/ Hif1b that is essential for Hif transcriptional function rescues the phenotype. Conversely, stabilization of HIF2A alone in podocytes results in crescentic glomerular disease. Together, our results show that the Hif pathway and Hif2a in particular are key players in maintenance of the glomerular barrier.

SummaryVascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.

PURPOSE OF REVIEW: This review discusses the recent evidence that intrinsic glomerular cells including podocytes, parietal epithelial cells and progenitor cells within Bowman's capsule contribute to cellular crescents.RECENT FINDINGS: Using a variety of newer molecular markers and lineage tracing experiments, investigators have clearly demonstrated that glomerular cells play a key role in the development and progression of cellular crescents in experimental and human disease.SUMMARY: Crescentic glomerulonephritis is associated with significant morbidity and mortality. Current therapies target the immune system. The recent finding that nonimmune cells also play a role in crescent formation highlights the need to identify alternate and complimentary therapeutic strategies.

The pathophysiology underlying the nephrotic syndrome is becoming clear for several inherited podocytopathies; the mechanisms of injury that lead to the acquired forms of this disease are not well understood. We explored these mechanisms using the mouse model of adriamycin-induced proteinuria.We estimated the fractional clearances for FITC-Ficolls, albumin, and neutral albumin in cooled, isolated,perfused kidneys (cIPK) in situ. Treatment with adriamycin led to a significant increase in the fractional clearance of albumin and of Ficoll with radii larger than 20 A. Neutral albumin (33.4 A) and similarly sized Ficoll behaved similarly to each other. In addition, adriamycin led to a significant loss of charge density and size selectivity of the glomerular barrier. The thickness of the glomerular endothelial surface layer(i.e., or the glycocalyx) in adriamycin-treated animals was only 20% of that in normal animals. Finally,several proteoglycans were downregulated in isolated glomeruli. In summary, adriamycin thins the glomerular glycocalyx, perhaps by downregulating proteoglycan synthesis, and alters glomerular charge- and size selectivity. These data suggest that the glomerular endothelium may play a role in the pathogenesis of proteinuric renal diseases.

Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.

The present study investigated whether sympathetic neurotransmission is altered at an early stage of diabetes in mesenteric small arteries isolated from female non-obese diabetic (NOD) and control animals without diabetes from the same mouse strain. The NOD diabetic mice had increased plasma glucose and hypertension. Confocal microscopy showed distribution of nerve terminals was similar, but immunoreaction intensity for neuropeptide Y (NPY) and tyrosine hydroxylase was higher in small arteries from NOD diabetic compared with NOD control mice. In the presence of prazosin and activated with vasopressin, electrical field stimulation evoked contractions which were more pronounced in mesenteric arteries from NOD diabetic versus NOD control mice and inhibited by the NPY Y(1) receptor antagonist, BIBP 3226. NPY concentration-response curves were leftward shifted in arteries from NOD diabetic versus NOD control both in arteries with and without endothelium, but not in the presence of the BIBP 3226. The present findings suggest that enhanced NPY content and vasoconstriction to NPY by activation of NPY Y(1) receptors in arteries from diabetic mice may contribute to the enhanced sympathetic nerve activity and vascular resistance in female non-obese early diabetic animals.

This is the first functional study of glomerular size and charge selectivity in mice. The aim was to investigate the controversial issue of glomerular permselectivity in animals exposed to glucosaminoglycan-degrading enzymes, hyaluronidase, and heparinase. Fractional clearances (theta) for FITC-Ficoll and albumin were estimated in isoflurane anesthetized mice in vivo and in cooled isolated perfused kidneys (cIPK). In cIPK, a significant increase of theta(albumin) from 0.0023 (95% confidence interval, 0.0014 to 0.0033) in controls to 0.0130 (95% confidence interval, 0.0055 to 0.0206) was seen after hyaluronidase treatment. The theta for neutral Ficoll of similar size as albumin was 0.063 to 0.093 in all groups. According to a heterogeneous charged fiber model, the fiber volume fraction of negatively charged fibers decreased by 10% after enzyme treatments. It is concluded that glomerular size and charge selectivity in mice is similar to that previously shown for rats. Moreover, hyaluronic acid, chondroitin sulfate, and heparan sulfate are of importance for charge selectivity.

SummaryDiabetic nephropathy (DN) is the leading cause of renal failure in the world. It is characterized by albuminuria and abnormal glomerular function and is considered a hyperglycemic “microvascular” complication of diabetes, implying a primary defect in the endothelium. However, we have previously shown that human podocytes have robust responses to insulin. To determine whether insulin signaling in podocytes affects glomerular function in vivo, we generated mice with specific deletion of the insulin receptor from their podocytes. These animals develop significant albuminuria together with histological features that recapitulate DN, but in a normoglycemic environment. Examination of “normal” insulin-responsive podocytes in vivo and in vitro demonstrates that insulin signals through the MAPK and PI3K pathways via the insulin receptor and directly remodels the actin cytoskeleton of this cell. Collectively, this work reveals the critical importance of podocyte insulin sensitivity for kidney function.