The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

Expression of the SARS-CoV-2 Receptor ACE2 in Human Retina and Diabetes-Implications for Retinopathy.

Purpose: To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. Methods: Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. Results: We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. Conclusions: Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.

Phosphorylation of pericyte FAK-Y861 affects tumour cell apoptosis and tumour blood vessel regression.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the specific involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using PdgfrßCre + ;FAKWT/WT, PdgfrßCre + ;FAKY397F/Y397F and PdgfrßCre + ;FAKY861F/Y861F mice, our data demonstrate that Lewis lung carcinoma tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrßCre + ;FAKY861F/Y861F but not PdgfrßCre + ;FAKY397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of pericyte derived signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.

Role of angiotensin-converting enzyme 2 and pericytes in cardiac complications of COVID-19 infection.

The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly reached pandemic proportions, and knowledge about this virus and coronavirus disease 2019 (COVID-19) has expanded rapidly. This review focuses primarily on mechanisms that contribute to acute cardiac injury and dysfunction, which are common in patients with severe disease. The etiology of cardiac injury is multifactorial, and the extent is likely enhanced by preexisting cardiovascular disease. Disruption of homeostatic mechanisms secondary to pulmonary pathology ranks high on the list, and there is growing evidence that direct infection of cardiac cells can occur. Angiotensin-converting enzyme 2 (ACE2) plays a central role in COVID-19 and is a necessary receptor for viral entry into human cells. ACE2 normally not only eliminates angiotensin II (Ang II) by converting it to Ang-(1-7) but also elicits a beneficial response profile counteracting that of Ang II. Molecular analyses of single nuclei from human hearts have shown that ACE2 is most highly expressed by pericytes. Given the important roles that pericytes have in the microvasculature, infection of these cells could compromise myocardial supply to meet metabolic demand. Furthermore, ACE2 activity is crucial for opposing adverse effects of locally generated Ang II, so virus-mediated internalization of ACE2 could exacerbate pathology by this mechanism. While the role of cardiac pericytes in acute heart injury by SARS-CoV-2 requires investigation, expression of ACE2 by these cells has broader implications for cardiac pathophysiology.

Recombinant Extracellular Domain (p75ECD) of the Neurotrophin Receptor p75 Attenuates Myocardial Ischemia-Reperfusion Injury by Inhibiting the p-JNK/Caspase-3 Signaling Pathway in Rat Microvascular Pericytes.

Background Pro-NTs (precursor of neurotrophins) and their receptor p75 are potential targets for preventing microvascular dysfunction induced by myocardial ischemia-reperfusion injury (IRI). p75ECD (ectodomain of neurotrophin receptor p75) may physiologically produce neurocytoprotective effects by scavenging pro-NTs. We therefore hypothesized that p75ECD may have a cardioprotective effect on IRI through microvascular mechanisms. Methods and Results Myocardial IRI was induced in Sprague-Dawley rats by occluding the left main coronary arteries for 45 minutes before a subsequent relaxation. Compared with the ischemia-reperfusion group, an intravenous injection of p75ECD (3 mg/kg) 5 minutes before reperfusion reduced the myocardial infarct area at 24 hours after reperfusion (by triphenyltetrazolium chloride, 44.9±3.9% versus 34.6±5.7%, P<0.05); improved the left ventricular ejection fraction (by echocardiography), with less myocardial fibrosis (by Masson's staining), and prevented microvascular dysfunction (by immunofluorescence) at 28 days after reperfusion; and reduced myocardial pro-NTs expression at 24 hours and 28 days after reperfusion (by Western blotting). A simulative IRI model using rat microvascular pericytes was established in vitro by hypoxia-reoxygenation (2/6 hours) combined with pro-NTs treatment (3 nmol/L) at R. p75ECD (3 µg/mL) given at R improved pericyte survival (by methyl thiazolyl tetrazolium assay) and attenuated apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling). In the reperfused hearts and hypoxia-reoxygenation +pro-NTs-injured pericytes, p75ECD inhibited the expression of p-JNK (phospho of c-Jun N-terminal kinase)/caspase-3 (by Western blotting). SP600125, an inhibitor of JNK, did not enhance the p75ECD-induced infarct-sparing effects and pericyte protection. Conclusions p75ECD may attenuate myocardial IRI via pro-NTs reduction-induced inhibition of p-JNK/caspase-3 pathway of microvascular pericytes in rats.

Redox signalling and regulation of the blood-brain barrier.

Neurological disorders are associated with increased oxidative stress. Reactive oxidants damage tissue and promote cell death, but it is apparent that oxidants can have more subtle effects on cell function through the modulation of redox-sensitive signalling pathways. Cells of the blood-brain barrier regulate the brain microenvironment but become dysfunctional during neurological disease. The blood-brain barrier is maintained by many cell types, and is modulated by redox-sensitive pathways, ranging from the cytoskeletal elements responsible for establishing a barrier, to growth factor and cytokine signalling pathways that influence neurovascular cells. During neurological disease, blood-brain barrier cells are exposed to exogenously generated oxidants from immune cells, as well as increasing endogenously oxidant production. These oxidants impair the function of the blood-brain barrier, leading to increased leakage and reduced blood flow. Reducing the impact of oxidants on the function of blood-brain barrier cells may provide new strategies for delaying the progression of neurological disease.

Phosphoinositide 3-Kinase-Regulated Pericyte Maturation Governs Vascular Remodeling.

BACKGROUND: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. METHODS: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kß isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. RESULTS: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kß, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kß inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. CONCLUSIONS: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kß activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kß activity.

The ACE2 expression in human heart indicates new potential mechanism of heart injury among patients infected with SARS-CoV-2.

A new type of pneumonia caused by a novel coronavirus SARS-CoV-2 outbreaks recently in China and spreads into many other countries. This disease, named as COVID-19, is similar to patients infected by SARS-CoV and MERS-CoV, and nearly 20% of patients developed severe condition. Cardiac injury is a prevalent complication of severe patients, exacerbating the disease severity in coronavirus disease 2019 (COVID-19) patients. Angiotensin-converting enzyme 2 (ACE2), the key host cellular receptor of SARS-CoV-2, has been identified in multiple organs, but its cellular distribution in human heart is not illuminated clearly. This study performed the first state-of-art single cell atlas of adult human heart, and revealed that pericytes with high expression of ACE2 might act as the target cardiac cell of SARS-CoV-2. The pericytes injury due to virus infection may result in capillary endothelial cells dysfunction, inducing microvascular dysfunction. And patients with basic heart failure disease showed increased ACE2 expression at both mRNA and protein levels, meaning that if infected by the virus these patients may have higher risk of heart attack and critically ill condition. The finding of this study explains the high rate of severe cases among COVID-19 patients with basic cardiovascular disease; and these results also perhaps provide important reference to clinical treatment of cardiac injury among severe patients infected by SARS-CoV-2.

A unique protein kinase C-dependent pathway for tissue factor downregulation in pericytes.

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11 h to 4.5 h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinase C (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes.

Brain alpha-amylase: a novel energy regulator important in Alzheimer disease?

Reduced glucose metabolism and formation of polyglucosan bodies (PGB) are, beside amyloid beta plaques and neurofibrillary tangles, well-known pathological findings associated with Alzheimer's disease (AD). Since both glucose availability and PGB are regulated by enzymatic degradation of glycogen, we hypothesize that dysfunctional glycogen degradation is a critical event in AD progression. We therefore investigated whether alpha (α)-amylase, an enzyme known to efficiently degrade polysaccharides in the gastrointestinal tract, is expressed in the hippocampal CA1/subiculum and if the expression is altered in AD patients. Using immunohistochemical staining techniques, we show the presence of the α-amylase isotypes AMY1A and AMY2A in neuronal dendritic spines, pericytes and astrocytes. Moreover, AD patients showed reduced gene expression of α-amylase, but conversely increased protein levels of α-amylase as well as increased activity of the enzyme compared with non-demented controls. Lastly, we observed increased, albeit not significant, load of periodic acid-Schiff positive PGB in the brain of AD patients, which correlated with increased α-amylase activity. These findings show that α-amylase is expressed and active in the human brain, and suggest the enzyme to be affected, alternatively play a role, in the neurodegenerative Alzheimer's disease pathology.

Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-ß, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca2+/calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Methylmercury induces hyaluronan synthesis in cultured human brain microvascular endothelial cells and pericytes via different mechanisms.

In a cerebrum damaged by methylmercury, where neuropathological lesions tend to localize along deep sulci and fissures, edematous changes in white matter have been proposed as the cause of such localization. Since hyaluronan has a high water-retention capability and can contribute to the progression of edematous changes, we hypothesize that methylmercury increases hyaluronan in brain microvascular cells. Our experimental results indicate that methylmercury induces the expression of hyaluronan in cultured human microvascular endothelial cells and pericytes through the induction of expressed UDP-glucose dehydrogenase and hyaluronan synthase 2, respectively. After exposure to methylmercury, hyaluronan largely accumulates in perivascular space, where it contributes to the progression of edematous changes.

Pericytes as Inducers of Rapid, Matrix Metalloproteinase-9-Dependent Capillary Damage during Ischemia.

Blood-brain barrier disruption (BBB) and release of toxic blood molecules into the brain contributes to neuronal injury during stroke and other cerebrovascular diseases. While pericytes are builders and custodians of the BBB in the normal brain, their impact on BBB integrity during ischemia remains unclear. We imaged pericyte-labeled transgenic mice with in vivo two-photon microscopy to examine the relationship between pericytes and blood plasma leakage during photothrombotic occlusion of cortical capillaries. Upon cessation of capillary flow, we observed that plasma leakage occurred with three times greater frequency in regions where pericyte somata adjoined the endothelium. Pericyte somata covered only 7% of the total capillary length in cortex, indicating that a disproportionate amount of leakage occurred from a small fraction of the capillary bed. Plasma leakage was preceded by rapid activation of matrix metalloproteinase (MMP) at pericyte somata, which was visualized at high resolution in vivo using a fluorescent probe for matrix metalloproteinase-2/9 activity, fluorescein isothiocyanate (FITC)-gelatin. Coinjection of an MMP-9 inhibitor, but not an MMP-2 inhibitor, reduced pericyte-associated FITC-gelatin fluorescence and plasma leakage. These results suggest that pericytes contribute to rapid and localized proteolytic degradation of the BBB during cerebral ischemia. SIGNIFICANCE STATEMENT: Pericytes are a key component of the neurovascular unit and are essential for normal BBB function. However, during acute ischemia, we find that pericytes are involved in creating rapid and heterogeneous BBB disruption in the capillary bed. The mechanism by which pericytes contribute to BBB damage warrants further investigation, as it may yield new therapeutic targets for acute stroke injury and other neurological diseases involving capillary flow impairment.

Regulation of high glucose-induced apoptosis of brain pericytes by mitochondrial CA VA: A specific target for prevention of diabetic cerebrovascular pathology.

Events responsible for cerebrovascular disease in diabetes are not fully understood. Pericyte loss is an early event that leads to endothelial cell death, microaneurysms, and cognitive impairment. A biochemical mechanism underlying pericyte loss is rapid respiration (oxidative metabolism of glucose). This escalation in respiration results from free influx of glucose into insulin-insensitive tissues in the face of high glucose levels in the blood. Rapid respiration generates superoxide, the precursor to all reactive oxygen species (ROS), and results in pericyte death. Respiration is regulated by carbonic anhydrases (CAs) VA and VB, the two isozymes expressed in mitochondria, and their pharmacologic inhibition with topiramate reduces respiration, ROS, and pericyte death. Topiramate inhibits both isozymes; therefore, in the earlier studies, their individual roles were not discerned. In a recent genetic study, we showed that mitochondrial CA VA plays a significant role in regulation of reactive oxygen species and pericyte death. The role of CA VB was not addressed. In this report, genetic knockdown and overexpression studies confirm that mitochondrial CA VA regulates respiration in pericytes, whereas mitochondrial CA VB does not contribute significantly. Identification of mitochondrial CA VA as a sole regulator of respiration provides a specific target to develop new drugs with fewer side effects that may be better tolerated and can protect the brain from diabetic injury. Since similar events occur in the capillary beds of other insulin-insensitive tissues such as the eye and kidney, these drugs may also slow the onset and progression of diabetic disease in these tissues.

Expression of CYP2R1 and VDR in human brain pericytes: the neurovascular vitamin D autocrine/paracrine model.

1,25 Dihydroxyvitamin D3 (1,25D) is a hormone produced from vitamin D through two hydroxylating steps catalyzed successively in the liver by the vitamin D 25-hydroxylase Cyp2R1 and in the kidney by the 25-hydroxyvitamin D3 1α-hydroxylase Cyp27B1. 1,25D behaves like a steroid hormone. It regulates gene transcription by interacting with a nuclear receptor named vitamin D receptor (VDR) for the vitamin D receptor. Although the role of vitamin D is historically related to rickets, its physiological function largely encompasses bone tissues. Accumulating evidence has indicated that 1,25D can also be considered a neurosteroid. For example, both VDR and CYP27B1 are expressed in brain cells. Similarly, the neuroprotective and anti-inflammatory potential of 1,25D in nervous tissue has been shown experimentally. The regulation of Cyp27B1, which catalyzes the last step of 1,25D synthesis, by the inflammatory cytokines tumor necrosis factor-α and interferon-γ has been reported recently. However, the fate of Cyp2R1 that catalyzes the first enzymatic reaction of the vitamin D metabolism has not received attention. Using human brain pericytes, we studied the expression of CYP2R1 and VDR genes when these cells were challenged to an inflammatory stimulus. We found a significant upregulation of these two genes in human brain pericytes challenged with tumor necrosis factor-α and interferon-γ. These results suggest the existence of an autocrine/paracrine vitamin D system in the neurovascular unit. The function of this novel signaling system might be critical in the control of neuroinflammation and in brain pathologies.

Modulación farmacológica de la cicatriz glial para la reparación de lesiones del SNC / Pharmacological modulation of the glial scar for CNS injury repair

Objetivo: Estudiar la actividad de la neurostatina obtenida enzimáticamente y purificada mediante un nuevo método, en cultivos de células implicadas en la formación de la cicatriz glial. Material y métodos: La neurostatina se obtuvo mediante una reacción enzimática a partir del gangliósido GD1b comercial y se purificó con un método nuevo simplificado. La actividad de la neurostatina se probó en ensayos de proliferación con MTT en pericitos y microglía de rata. Resultados: La actividad de la neurostatina purificada fue similar a la obtenida mediante purificación a partir de cerebro de mamífero. La neurostatina inhibió la proliferación de los pericitos inducida por el factor de crecimiento PDGF-B y la proliferación de la microglía de rata inducida por la toxina bacteriana LPS a concentraciones nanomolar. Conclusión: La nueva metodología de obtención y purificación de la neurostatina y su actividad justifican su ensayo en modelos de lesión del SNC en animales, para evaluar su posible uso como terapia en pacientes con lesiones del SNC (AU)

Objective: To study the activity of neurostatin obtained enzymatically and purified using a new method, in cultures of cells involved in the formation of the glial scar. Material and methods: Neurostatin was obtained from the commercial ganglioside GD1b, using enzymatic Oacetylation, and was purified by a new simplified method. The activity of neurostatin was tested by an MTT proliferation assay in pericytes and rat microglial cells. Results: The activity of neurostatin obtained and purified by this new method was similar to the neurostatin obtained by the purification from mammalian brain. Neurostatin inhibited the PDGF-B growth factor induced proliferation of pericytes and LPS bacteria toxin induced proliferation of rat microglial cells at nanomolar concentrations. Conclusion: This new methodology to synthesize and purify neurostatin and its activity justify further studies to test its effect in animal models of CNS injuries and to evaluate its possible use as a therapy in patients with CNS injuries (AU)

The involvement of high mobility group 1 cytokine and phospholipases A2 in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy, the main microvascular complications of diabetes and one of the leading causes of blindness worldwide. Interesting reports on the role of inflammatory/proangiogenic high mobility group 1 (HMGB-1) cytokine and phospholipases A2 (PLA2) in neovascularization have diverted our concentration to reveal whether HMGB-1 and PLA2 plays role in diabetic retinopathy. METHODS: We performed our study in streptozotocin (STZ)-induced diabetic rat model. The expression levels of the cytokines, chemokines, and cell adhesion molecules in retinal tissues were evaluated by quantitative RT-PCR. HMGB-1 and PLA2 protein levels along with VEGF, TNF-α, IL-1ß and ICAM-1 levels were also measured. RESULTS: We observed the retinal pericytes, endothelial injury/death and breakdown of blood-retinal barrier (BRB). The protein expression of HMGB-1, PLA2 and IL-1ß were significantly increased in micro vessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNF-α. Further investigation revealed that pericyte death is mediated by HMGB-1-induced cytotoxic activity of glial cells, while HMGB-1 can directly mediate endothelial cell death. Similarly, increased expression of PLA2 represents the diabetic mediated alteration of BRB, perhaps up regulating the VEGF. CONCLUSIONS: Our data suggest that HMGB-1 and PLA2 involved in retinal pericyte and endothelial injury and cell death in diabetic retinopathy. From this study, we suggest that HMGB-1 and PLA2 may be interesting targets in managing diabetic retinopathy.

Trkb signaling in pericytes is required for cardiac microvessel stabilization.

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

Endothelial PKCα-MAPK/ERK-phospholipase A2 pathway activation as a response of glioma in a triple culture model. A new role for pericytes?

In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumor and cells constituting microvessel, a triple culture model in which endothelial cells (EC), pericytes (PC) and glioma C6 cells were cultured together. In the present work, we observed that C6 enhanced EC proliferation. This effect was reduced by cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2), cyclooxygenase-2 (COX-2), PI3-K, MEK-1, and ERK1/2 inhibitors and by siRNAs against both PLA2s. In EC, C6 induced an increase in iPLA2, cPLA2 and COX-2 total protein expression. Moreover, the increase in endothelial cPLA2 phosphorylation was attenuated by kinase inhibitors. Both EC proliferation and signal protein phosphorylation were attenuated when PC were in triple culture. In EC/C6 supernatants, and, in a lesser extent, in EC/PC co-cultures, an enhancement in prostaglandins E2 (PGE2) was found. The presence of PC in triple-cultures caused a decrease in production of PGE2 respect to EC/C6 double-cultures. In all systems, AACOCF3 and BEL significantly reduced PGE2 secretion. In Matrigel-based assays, emerging branch points from EC cell bodies and tubule-like structures were observed. C6 conditioned EC/PC co-cultures in constituting poorly organized tubules. Transfection of EC with c- and iPLA2 siRNA strongly reduced in vitro tubulogenesis. Data here reported indicate that PKCα, ERK kinase phosphorylation, PLA2s and COX-2 activation, and PGE2 production in EC stimulated by tumor cells are coincident phenomena and could represent therapeutic targets in chemoprevention of glioma. Moreover, PC exhibited an important "modulating" role in the initial stages of angiogenesis driven by a brain tumor.

Nox4 is a major source of superoxide production in human brain pericytes.

BACKGROUND: Pericytes are multifunctional cells surrounding capillaries and postcapillary venules. In brain microvasculature, pericytes play a pivotal role under physiological and pathological conditions by producing reactive oxygen species (ROS). The aims of this study were to elucidate the source of ROS and its regulation in human brain pericytes. METHODS: The expression of Nox enzymes in the cells was evaluated using RT-PCR and western blot. Superoxide production was determined by superoxide dismutase-inhibitable chemiluminescence. Silencing of Nox4 was performed using RNAi, and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: Nox4 was predominant among the Nox family in human brain pericytes. Membrane fractions of cells produced superoxide in the presence of NAD(P)H. Superoxide production was almost abolished with diphenileneiodonium, a Nox inhibitor; however, inhibitors of other possible superoxide-producing enzymes had no effect on NAD(P)H-dependent superoxide production. Pericytes expressed angiotensin II (Ang II) receptors, and Ang II upregulated Nox4 expression. Hypoxic conditions also increased the Nox4 expression. Silencing of Nox4 significantly reduced ROS production and attenuated cell proliferation. CONCLUSION: Our study showed that Nox4 is a major superoxide-producing enzyme and that its expression is regulated by Ang II and hypoxic stress in human brain pericytes. In addition, Nox4 may promote cell growth.