Alterations In Endothelial Cell Function Research Articles

Nilotinib-induced alterations in endothelial cell function recapitulate clinical vascular phenotypes independent of ABL1

Nilotinib is a highly effective treatment for chronic myeloid leukemia but has been consistently associated with the development of nilotinib-induced arterial disease (NAD) in a subset of patients. To date, which cell types mediate this effect and whether NAD results from on-target mechanisms is unknown. We utilized human induced pluripotent stem cells (hiPSCs) to generate endothelial cells and vascular smooth muscle cells for in vitro study of NAD. We found that nilotinib adversely affects endothelial proliferation and migration, in addition to increasing intracellular nitric oxide. Nilotinib did not alter endothelial barrier function or lipid uptake. No effect of nilotinib was observed in vascular smooth muscle cells, suggesting that NAD is primarily mediated through endothelial cells. To evaluate whether NAD results from enhanced inhibition of ABL1, we generated multiple ABL1 knockout lines. The effects of nilotinib remained unchanged in the absence of ABL1, suggesting that NAD results from off- rather than on-target signaling. The model established in the present study can be applied to future mechanistic and patient-specific pharmacogenomic studies.

Single nuclei transcriptomics in diabetic mice reveals altered brain hippocampal endothelial cell function, permeability, and behavior

Type 2 diabetes mellitus (T2DM) is a metabolic disorder with cerebrovascular and cardiovascular sequelae. Yet, a clear pattern of gene dysregulation by T2DM in dementia has yet to be defined. We used single nuclei RNA sequencing technology to profile the transcriptome of endothelial cells (EC) from anatomically defined hippocampus of db/db mice to identify differentially expressed (DE) genes, gene pathways and networks, predicted regulating transcription factors, and targets of DE long noncoding RNAs. We also applied gadolinium (Gd) enhanced magnetic resonance imaging (MRI) to assess blood brain barrier (BBB) permeability, and functionally assessed cognitive behavior. The murine gene expression profiles were then integrated with those of persons with Alzheimer's disease (AD) and vascular dementia (VaD).We reveal that the transcriptome of the diabetic hippocampal murine brain endothelium differs substantially from control wild types with molecular changes characterized by differential RNA coding and noncoding pathways enriched for EC signaling and for endothelial functions for neuroinflammation, endothelial barrier disruption, and neurodegeneration. Gd enhanced structural brain MRI linked endothelial molecular alterations to BBB dysfunction by neuroimaging. Integrated multiomics of hippocampal endothelial gene dysregulation associated with impairments in cognitive adaptive capacity. In addition, the diabetic transcriptome significantly and positively correlated with that of persons with AD and VaD. Taken together, our results from comprehensive, multilevel, integrated, single nuclei transcriptomics support the hypothesis of T2DM-mediated neuroinflammation and endothelial cell and barrier disruption as key mechanisms in cognitive decline in T2DM, thereby suggesting potential endothelial-specific molecular therapeutic targets.

Pentastatin, a matrikine of the Collagen IVa5, is a novel endogenous mediator of pulmonary endothelial dysfunction.

Deposition of basement membrane components, such as collagen IVα5, is associated with altered endothelial cell function in pulmonary hypertension. Collagen IVα5 harbors a functionally active fragment within its C-terminal non-collageneous (NC1) domain, called pentastatin, whose role in pulmonary endothelial cell behavior remains unknown. Here, we demonstrate that pentastatin serves as a mediator of pulmonary endothelial cell dysfunction, contributing to pulmonary hypertension. In vitro, treatment with pentastatin induced transcription of immediate early genes and pro-inflammatory cytokines and led to a functional loss of endothelial barrier integrity in pulmonary arterial endothelial cells. Mechanistically, pentastatin leads to β1-integrin subunit clustering and Rho/ROCK activation. Blockage of the β1-integrin subunit or the Rho/ROCK pathway partially attenuated the pentastatin-induced endothelial barrier disruption. While pentastatin reduced the viability of endothelial cells, smooth muscle cell proliferation was induced. These effects on the pulmonary vascular cells were recapitulated ex vivo in the isolated-perfused lung model, where treatment with pentastatin induced swelling of the endothelium accompanied by occasional endothelial cell apoptosis. This was reflected by increased vascular permeability and elevated pulmonary arterial pressure induced by pentastatin. This study identifies pentastatin as a mediator of endothelial cell dysfunction, which thus might contribute to the pathogenesis of pulmonary vascular disorders such as pulmonary hypertension.

MicroRNA-150-5p is upregulated in the brain microvasculature during prenatal alcohol exposure and inhibits the angiogenic factor Vezf1.

Fetal alcohol spectrum disorders (FASD) occur in children who were exposed to alcohol in utero and are manifested in a wide range of neurocognitive deficits. These deficits could be caused by alterations to the cortical microvasculature that are controlled by post-transcriptional regulators such as microRNAs. Using an established mouse model of moderate prenatal alcohol exposure (PAE), we isolated cortices (CTX) and brain microvascular endothelial cells (BMVECs) at embryonic day 18 (E18) and examined the expression of miR-150-5p and potential downstream targets. Cellular transfections and intrauterine injections with LNA™ mimics or inhibitors were used to test miR-150-5p regulation of novel target vascular endothelial zinc finger 1 (Vezf1). Dual-luciferase assays were used to assess the direct binding of miR-150-5p to the Vezf1 3'UTR. The effects of miR-150-5p and Vezf1 on endothelial cell function were determined by in vitro migration and tube formation assays. We found that miR-150-5p was upregulated and Vezf1 was downregulated during PAE in the E18 CTX and BMVECs. Transfection with miR-150-5p mimics resulted in decreased Vezf1 expression in BMVECs, while miR-150-5p inhibition did the opposite. Dual-luciferase assays revealed direct binding of miR-150-5p with the Vezf1 3'UTR. Intrauterine injections showed that miR-150-5p regulates the expression of Vezf1 in vivo during PAE. miR-150-5p overexpression decreased BMVEC migration and tube formation, while miR-150-5p inhibition enhanced migration and tube formation. Vezf1 overexpression rescued the effects of the miR-150-5p mimic. Alcohol treatment of BMVECs increased miR-150-5p expression and inhibited migration and tube formation. Finally, miR-150-5p inhibition and Vezf1 overexpression rescued the negative effects of alcohol on migration and tube formation. miR-150-5p regulation of Vezf1 results in altered endothelial cell function during alcohol exposure. Further, miR-150-5p inhibition of Vezf1 may adversely alter the development of the cortical microvasculature during PAE and contribute to deficits seen in patients with FASD.

PD146176 affects human EA.hy926 endothelial cell function by differentially modulating oxylipin production of LOX, COX and CYP epoxygenase

Oxylipins are oxygenated derivatives of polyunsaturated fatty acids, generated by COX, LOX and CYP enzymes, that regulate various aspects of endothelial cell physiology. Although 15-LOX and its products are positively associated with atherosclerosis, the relevant mechanisms have not been explored. The current study examined the effects of PD146176 (PD), a putative 15-LOX inhibitor, on EA.hy926 endothelial cell functions in the growing and confluent states. The effects of PD on endothelial cell oxylipin production (profiled by LC/MS/MS), cell viability, proliferation, eNOS activity, ICAM-1 and VE-cadherin levels were assessed. The contribution of signaling pathways relevant to endothelial function (p38 MAPK, Akt, PPARα) were also investigated. PD treatment for 30 min did not block formation of individual 15-LOX oxylipins, but 20 μM PD stimulated the accumulation of total LOX and COX products, while reducing several individual CYP products generated by epoxygenase. At 20 μM, the accumulated total oxylipins were primarily LOX-derived (86%) followed by COX (12%) and CYP (2%). PD altered cell functions by upregulating p38 MAPK and PPARα and downregulating Akt in a dose-dependent fashion. These observations suggest a link between PD-induced changes in oxylipins and altered endothelial cell functions via specific signaling pathways. In conclusion, the results of this study imply that PD does not function as a 15-LOX inhibitor in EA.hy926 endothelial cells, and instead inhibits CYP epoxygenase. These findings suggest that the cellular function changes induced by PD may be contingent upon its ability to modulate total oxylipin production, particularly by the LOX and CYP families.

COVID-19 during pregnancy one year on - what lessons did we learn?

It is now more than a year since the first case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) was diagnosed in China. Current data suggest that pregnancy may not only be a risk factor for the development of severe forms of COVID-19, but that the SARS-CoV-2 infection may impact on common pregnancy complications as well. Healthy pregnant women are likely to be more susceptible to viral infection and therefore are at higher risk of developing severe COVID-19 because of adaptive changes in their immune and respiratory systems, their altered endothelial cell functions, and modified coagulation responses. However, studies show that most pregnant women diagnosed with COVID-19 developed mild-to-moderate symptoms and only a few of them have required critical care facilities. In contrast with preeclampsia, preeclampsia-like syndrome can resolve spontaneously following recovery from severe pneumonia and may not be an obstetric indication for delivery. Preeclampsia-like syndrome is one symptom of COVID-19, but its cause is different from obstetric preeclampsia and therefore not connected with placental failure. Vertical transmission of SARS-CoV-2 infection is rare but can probably occur. No evidence has been found that COVID-19 developed during pregnancy leads to unfavourable outcomes in the fetus. Most health authorities indicate that standard procedures should be used when managing pregnancy complications in asymptomatic women with confirmed SARS-CoV-2. Vaccines should not be withheld from pregnant and lactating individuals who otherwise meet the vaccination criteria.

Manipulating niche composition limits damage to haematopoietic stem cells during Plasmodium infection.

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and-coupled with reactive oxygen species quenching-enables partial rescuing of HSC function.

Basement Membrane Remodeling Controls Endothelial Function in Idiopathic Pulmonary Arterial Hypertension.

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.

Analysis of cerebrovascular dysfunction caused by chronic social defeat in mice

Psychological stress and affective disorders are clinically associated with hypertension and vascular disease, but the biological links between the conditions have not been fully explored. To examine this relationship, we used chronic social defeat (CSD) stress, which produces anxiety-like and depressive-like behavioral declines in susceptible mice. In such mice, CSD also produces cerebrovascular microbleeds in scattered locations. Here, we showed further evidence of vascular pathology and blood–brain barrier breakdown by visualizing plasma immunoglobulins and erythrocytes within the parenchyma and perivascular spaces of CSD brains. To further characterize the impact of stress on the cerebrovasculature, brain endothelial cells (bECs) were isolated, and global gene expression profiles were generated. Bioinformatic analysis of CSD-induced transcriptional changes in bECs showed enrichment in pathways that delineate the vascular response to injury. These pathways followed a temporal sequence of inflammation, oxidative stress, growth factor signaling, and wound healing (i.e., platelet aggregation, hemostasis, fibrinogen deposition, and angiogenesis). Immunohistochemical staining for markers of fibrinogen deposition and angiogenesis confirmed the existence of the markers at the sites of vascular disruptions. Recovery after CSD cessation was marked by recruitment of leukocytes perhaps participating in vascular repair. The data suggest that co-morbidity of affective disorders and vascular diseases may be attributed in part to a common link in altered endothelial cell function.

Assessment of serum Lactate Dehydrogenase in preeclamptic pregnant women

Background: Evidences prove that endothelial cell and altered endothelial cell function play an important role in the pathogenesis of preeclampsia. Therefore serum lactate dehydrogenase (LDH) is useful biochemical markers reflecting the severity of the occurrence of preeclampsia. Objective: Determination of serum lactate dehydrogenase (LDH) in severe preeclamptic pregnant women. Methods: This is the case-control hospital based study carried in the Al Azhar Medical College and Super speciality Hospital, Thodupuzha. Normal pregnant women (n=55) and women with severe preeclampsia (n=63) were included in the study. Both the groups were in their third trimester and of same age and same gestational age. Serum LDH levels were measured by continuous spectrophotometric pyruvate method for both groups. Results: Higher values of serum lactate dehydrogenase (LDH) were found in severe preeclamptic women compared with normal pregnant women in third trimester. Conclusion: Elevated levels of serum LDH indicates the tissue damage and might be the cause of the occurrence of preeclampsia.

Vascular smooth muscle cell dysfunction in diabetes: nuclear receptors channel to relaxation.

Endothelial dysfunction and impaired vascular relaxation represent a common cause of microvascular disease in patients with diabetes. Although multiple mechanisms underlying altered endothelial cell function in diabetes have been described, there is currently no specific and approved pharmacological treatment. In this edition of Clinical Science, Morales-Cano et al. characterize voltage-dependent K(+) (Kv) channels as genes regulated by pharmacological activation of peroxisome proliferator-activated receptor-b/d (PPARb/d). Diabetes altered Kv channel function leading to impaired coronary artery relaxation, which was prevented by pharmacological activation of PPARb/d. These studies highlight an important mechanism of vascular dysfunction in diabetes and point to a potential approach for therapy, particularly considering that PPARb/d ligands have been developed and tested in small clinical trials.

Role of the endothelial glycocalyx in kidney disease and vascular dysfunction

BackgroundAlbuminuria, an early marker of kidney disease, is strongly and independently associated with adverse cardiovascular outcomes, including heart attacks and strokes. Nonetheless, the mechanisms linking albuminuria and adverse cardiovascular events remain unclear. The endothelial glycocalyx, a friable but protective lining of all blood vessels, is damaged in both atherosclerosis and kidney disease. We examined whether factors in the plasma of patients with kidney disease could damage the endothelial glycocalyx, and sought underlying mechanisms. MethodsMetabolic labelling of glycosaminoglycans (GAGs) was achieved by culturing human glomerular endothelial cells in media containing 20 μCi/mL D-[3H3] glucosamine for 24 h. Monolayers were washed and subsequently exposed either to conditioned serum free media containing 10% plasma from patients with either heavy albuminuria (from an intrinsic renal disease) or from healthy controls for 24h, or to 100 μM hydrogen peroxide or vehicle for 4 h. The supernatant was then collected and applied to a di-ethyl-amino-ethyl fast flow column connected to a fast protein liquid chromatography system. Samples were applied to the column at 1 mL/min and subsequently merged with column matrix for 15 min. Bound proteoglycans were eluted with 7M urea (pH 6) and collected in 1 mL fractions. All fractions were quantified by liquid scintillation counting. FindingsExposure of glomerular endothelial cells to plasma from three patients with albuminuria resulted in reduced levels of GAGs into the media (control plasma 112 514 disintegrations per min (DPM) [SE 6153], albuminuric plasma 74 743 DPM [9607]; p<0·05 unpaired t test), indicating altered endothelial cell GAG metabolism that might represent decreased turnover. Because oxidative stress is regarded as a key mediator of endothelial dysfunction in kidney disease, we generated reactive oxygen species by exposing cells to hydrogen peroxide. This exposure resulted in a different pattern of GAG turnover: shedding of radiolabelled GAGs into the media was increased (vehicle 1302 [684] DPM D-[3H3] glucosamine-labelled heparan sulphate GAG, hydrogen peroxide 25 053 [6635] DPM D-[3H3] glucosamine-labelled heparan sulphate GAG; p<0·005 unpaired t test). InterpretationDamage to the endothelial glycocalyx in response to factors in albuminuric plasma provides a novel concept in understanding endothelial dysfunction in disease states. This work provides evidence that the albuminuric state itself might lead to systemic alterations in endothelial cell function, although our preliminary studies indicate that reactive oxygen species are unlikely to be involved in these changes. We are now seeking alternative pathways underlying the effects of albuminuric plasma on the endothelial glycocalyx, assessing whether the endothelial glycocalyx is damaged in blood vessels in situ in patients with kidney disease, and examining whether the endothelial glycocalyx can be restored (and endothelial cell function improved) by manipulating these pathways. Our long-term goal is to manipulate endothelial glycocalyx regulation to develop an entirely new class of treatments for vasculoprotection in kidney disease and other conditions. FundingNational Institute for Health Research.

Wall morphology, blood flow and wall shear stress: MR findings in patients with peripheral artery disease

To investigate the influence of atherosclerotic plaques on femoral haemodynamics assessed by two-dimensional (2D) phase-contrast (PC) magnetic resonance imaging (MRI) with three-directional velocity encoding. During 1year, patients with peripheral artery disease and an ankle brachial index <1.00 were enrolled. After institutional review board approval and written informed consent, 44 patients (age, 70 ± 12years) underwent common femoral artery MRI. Patients with contra-indications for MRI were excluded. Sequences included 2D time-of-flight, proton-density, T1-weighted and T2-weighted MRI. Electrocardiogram (ECG)-gated 2D PC-MRI with 3D velocity encoding was acquired. A radiologist classified images in five categories. Blood flow, velocity and wall shear stress (WSS) along the vessel circumference were quantified from the PC-MRI data. The acquired images were of good quality for interpretation. There were no image quality problems related to poor ECG-gating or slice positioning. Velocities, oscillatory shear stress and total flow were similar between patients with normal arteries and wall thickening/plaque. Patients with plaques demonstrated regionally increased peak systolic WSS and enhanced WSS eccentricity. Combined multi-contrast morphological imaging of the peripheral arterial wall with PC-MRI with three-directional velocity encoding is a feasible technique. Further study is needed to determine whether flow is an appropriate marker for altered endothelial cell function, vascular remodelling and plaque progression. • Femoral plaques are associated with altered dynamics of peripheral blood flow. • Multi-contrast MRI can investigate the presence and type of atherosclerotic plaques. • Three-dimensional velocity-encoding phase-contrast MRI can investigate flow and wall shear stress. • Atherosclerotic peripheral arteries demonstrate increased systolic velocities and wall shear stress.

Oxidative Stress and Pathological Changes After Coronary Artery Interventions

Oxidative stress greatly influences the pathogenesis of various cardiovascular disorders. Coronary interventions, including balloon angioplasty and coronary stent implantation, are associated with increased vascular levels of reactive oxygen species in conjunction with altered endothelial cell and smooth muscle cell function. These alterations potentially lead to restenosis, thrombosis, or endothelial dysfunction in the treated artery. Therefore, the understanding of the pathophysiological role of reactive oxygen species (ROS) generated during or after coronary interventions, or both, is essential to improve the success rate of these procedures. Superoxide O2(·-) anions, whether derived from uncoupled endothelial nitric oxide synthase, nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase, or mitochondria, are among the most harmful ROS. O2(·-) can scavenge nitric oxide, modify proteins and nucleotides, and induce proinflammatory signaling, which may lead to greater ROS production. Current innovations in stent technologies, including biodegradable stents, nitric oxide donor-coated stents, and a new generation of drug-eluting stents, therefore address persistent oxidative stress and reduced nitric oxide bioavailability after percutaneous coronary interventions. This review discusses the molecular mechanisms of ROS generation after coronary interventions, the related pathological events-including restenosis, endothelial dysfunction, and stent thrombosis-and possible therapeutic ways forward.

Effect of α,β-unsaturated aldehydes on endothelial cell growth in bacterial cellulose for vascular tissue engineering

Cardiovascular disease is the leading global cause of death. Cigarette smoking is a major risk factor for cardiovascular disease. And α,β-unsaturated aldehydes such as acrolein and crotonaldehyde are major component of cigarette smoke. Use of replacement grafts materials is one of the cardiovascular disease treatments. However, currently available synthetic materials generally produce poor outcomes including hyperplasia and thrombogenicity. Recently, bacterial synthesized cellulose has received interest as a new functional vascular graft biomaterial owing to its biocompatibility. However, the association of a bacterial cellulose-based scaffold and cigarette smoke is not known. The present study investigated the alteration of function of human umbilical vein endothelial cells (HUVECs) treated with α,β-unsaturated aldehyde on bacterial cellulose-based material. The data suggest that α,β-unsaturated aldehydes in cigarette smoke induce altered endothelial cell functions including morphology, adhesion, proliferation, viability and growth on bacterial cellulose. These results may provide the view that cigarette smoking of cardiovascular disease patients applied to bacterial cellulose-based vascular grafts is risk.

Increased transvascular transport of WGA-peroxidase after chronic perinatal stress in the hippocampal microvasculature of the rat

Brain endothelial ultrastructural properties contribute to maintain proper blood–brain barrier (BBB) function. Several physiological and pathological conditions have been shown to alter BBB permeability to blood-borne molecules, acute and chronic stress among them. In the rat, early life stress increased transvascular transport of Evans blue, however, the route of tracer extravasation is not fully known; therefore the aim of the present experiment was to describe the ultrastructural changes in endothelial cells subsequent to chronic perinatal stress in order to ascertain the route for transvascular transport of an electrodense tracer. Pregnant Wistar rats and their litters were used. Four pregnant rats were subjected to forced swimming between gestational days 10 to 20. After delivery, half of the control litters underwent 180 min maternal separation from postnatal day 2 to 20. Controls were kept free of any stress manipulation. At sacrifice between postnatal days 1 to 30 subjects were given intracardially the lectin wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). WGA-HRP stained hippocampi were processed for ultrastructural analysis, transmission electron micrographs were obtained and endothelial ultrastructural parameters quantified using the ImageJ software. Both stress procedures accelerated gross microvessel development by decreasing capillary wall thickness and endothelial microvilli. However, early-life stress also neutralized endothelial glycocalyx, increased vesicle-mediated transport and tended to promote the formation of secondary lysosomes containing endocytosed WGA-HRP vesicles, all parameters of altered endothelial cell function. Tight junction development in both stress groups was similar to the control pups.

Risk Factors for Corneal Infiltrative Events in Soft Contact Lens (SCL) Wearers: A Case Control Study in 2010

Radiation-induced endothelial/vascular injury is a major complicating factor in radiotherapy and a leading cause of morbidity and mortality in nuclear or radiological catastrophes. Exposure of tissue to ionizing radiation (IR) leads to the release of oxygen radicals and proteases that result in loss of endothelial barrier function and leukocyte dysfunction leading to tissue injury and organ damage. Microvascular endothelial cells are particularly sensitive to IR and radiation-induced alterations in endothelial cell function are thought to be a critical factor in organ damage through endothelial cell activation, enhanced leukocyte-endothelial cell interactions, increased barrier permeability and initiation of apoptotic pathways. These radiation-induced inflammatory responses are important in early and late radiation pathologies in various organs. A better understanding of mechanisms of radiation-induced endothelium dysfunction is therefore vital, as radiobiological response of endothelium is of major importance for medical management and therapeutic development for radiation injuries. In this review, we summarize the current knowledge of cellular and molecular mechanisms of radiation-induced endothelium damage and their impact on early and late radiation injury. Furthermore, we review established and emerging in vivo and in vitro models that have been developed to study the mechanisms of radiation-induced endothelium damage and to design, develop and rapidly screen therapeutics for treatment of radiation-induced vascular damage. Currently there are no specific therapeutics available to protect against radiation-induced loss of endothelial barrier function, leukocyte dysfunction and resulting organ damage. Developing therapeutics to prevent endothelium dysfunction and normal tissue damage during radiotherapy can serve as the urgently needed medical countermeasures.

Hypoglycemia and Vascular Disease

Macrovascular and microvascular complications commonly occur in individuals with type 1 and type 2 diabetes. The mechanisms responsible for these complications remain the subjects of intense investigation. With regard to atherosclerosis specifically, there have been great strides in describing the pathophysiological connection between alterations in the diabetic metabolic state and altered endothelial, platelet, and vascular smooth muscle cell functions. Hyperglycemia and insulin resistance independently disrupt nitric oxide synthesis and signaling, induce the production of reactive oxygen species, and up-regulate inflammatory and thrombotic markers and mediators of vasoconstriction (1, 2). Conversely, insulin per se has been shown to actually have vasodilatory and antiinflammatory effects independent of glycemia. Insulin exerts these beneficial effects via increased nitric oxide synthase production (thereby causing increased nitric oxide synthesis) and the suppression of reactive oxygen species, inflammatory mediators, and thrombotic markers (2). To overcome the deleterious effects of hyperglycemia and insulin resistance, clinicians have increasingly used intensive glucose control for both type 1 and type 2 diabetes. Unfortunately, as glycemic control improves, rates of hypoglycemia increase. Multicenter randomized controlled trials to examine the effects of stricter glycemic control in both type 1 and type 2 diabetes have confirmed that not only do the total rates of hypoglycemia increase with improved glycemia, but the incidence of severe disabling hypoglycemia also increases. Two recent large studies on glucose control and complications in type 2 diabetes mellitus [the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Trial and the Veterans Affairs Diabetes Trial] found no benefit of improved glycemic control on macrovascular complications. In fact, the ACCORD study found an increased mortality rate in the intensive glucose–control arm of the study, which was stopped earlier than planned. A major question that emanated from these studies was why intensive glycemic control did not produce beneficial effects. One …

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

BackgroundIncreased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.Methods and FindingsHere, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.ConclusionsThese results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.

Pathogenic Hantaviruses Direct the Adherence of Quiescent Platelets to Infected Endothelial Cells

Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of beta(3) integrins in hemostasis and the inactivation of beta(3) integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell beta(3) integrins and neutralized by the addition of the anti-beta(3) Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and recruitment to vast endothelial cell beds further suggests mechanisms by which hantaviruses may cause thrombocytopenia and induce hypoxia. These findings are fundamental to our understanding of pathogenic-hantavirus regulation of endothelial cell responses that contribute to vascular permeability.