Microvessel Rarefaction Research Articles

Abstract P334: Impairment Of Cognitive Ability In Middle-aged Renin Hypertensive Transgene Mice

Background: Vascular dementia (VD) is a severe cognitive impairment syndrome, characterized by a progressive memory and cognitive skill decline. Approximately 40 million people have dementia worldwide, and this number is expected to increase up to 115 million by 2050 due to the aging population. To date, no proven interventions are available to prevent VD. Chronic exposure to risk factors for vascular diseases, such as hypertension, diabetes, are implicated in the pathogenesis of VD, but the mechanisms remain to be fully understood. Methods: In this study, we utilized a renin hypertensive transgene mice (R+) mouse model to explore how the elevated blood pressure affects the pathogenesis of VD. We analyzed the cognitive ability, cerebral microvascular density, and neurons in young R+ mice, middle-aged R+ mice, and the wild type (WT) mice. Results: We found that middle-aged R+ mice had increased blood pressure associated with cognitive impairment as revealed by prolonged latency to target in morris water maze as compared to the young R+ mice and age-matched WT mice did. Meanwhile, our data showed that the R+ mice had brain microvessel rarefaction in the cortex and hippocampus, associating with a significantly low number of neurons in the hippocampus region (subiculum) as compared to that in WT mice. Conclusion: Our data have demonstrated that middle-aged renin transgene mice have impaired cognitive ability, accompanying with a low density of microvessel and fewer neurons, suggesting that chronic cerebral hypoperfusion might further damage neuron which could contribute to the progress of VD.

Microvascular Dysfunction in Skeletal Muscle Precedes Myocardial Vascular Changes in Diabetic Cardiomyopathy: Sex-Dependent Differences.

AimTo uncover sex-related microvascular abnormalities that underlie the early presentation of reduced perfusion in leg skeletal muscle in a type II rat model of diabetic cardiomyopathy.Methods and ResultsDiabetes was induced using a non-obese, diet-based, low-dose streptozotocin model in adult female (18 diabetic, 9 control) and male rats (29 diabetic, 11 control). Time-course monitoring over 12 months following diabetes induction was performed using echocardiography, treadmill exercise, photoacoustic imaging, flow-mediated dilation (FMD), histopathology, and immunohistochemistry. Diabetic rats maintained normal weights. Hypertension appeared late in both diabetic males (7 months) and females (10 months), while only diabetic males had elevated cholesterol (7 months). On echocardiography, all diabetic animals maintained normal ejection fraction and exhibited diastolic dysfunction, mild systolic dysfunction, and a slightly enlarged left ventricle. Exercise tolerance declined progressively and early in males (4 months), later in females (8 months); FMD showed lower baseline femoral arterial flow but unchanged reactivity in both sexes (5 months); and photoacoustic imaging showed lower tissue oxygen saturation in the legs of diabetic males (4 months) and diabetic females (10 months). Myocardial perfusion was normal in both sexes. Histopathology at the final timepoint of Month 10 (males) and Month 12 (females) revealed that myocardial microvasculature was normal in both vessel density and structure, thus explaining normal perfusion on imaging. However, leg muscle microvasculature exhibited perivascular smooth muscle thickening around small arterioles in diabetic females and around large arterioles in diabetic males, explaining the depressed readings on photoacoustic and FMD. Histology also confirmed the absence of commonly reported HFpEF markers, including microvessel rarefaction, myocardial fibrosis, and left ventricular hypertrophy.ConclusionExercise intolerance manifesting early in the progression of diabetic cardiomyopathy can be attributed to decreased perfusion to the leg skeletal muscle due to perivascular smooth muscle thickening around small arterioles in females and large arterioles in males. This microvascular abnormality was absent in the myocardium, where perfusion levels remained normal throughout the study. We conclude that although skeletal muscle microvascular dysfunction of the vasculature presents at different levels depending on sex, it consistently presents early in both sexes prior to overt cardiac changes such as rarefaction, fibrosis, or hypertrophy.

VEGF Receptor Inhibitor-Induced Hypertension: Emerging Mechanisms and Clinical Implications.

While vascular endothelial growth factor receptor inhibitors (VEGFRis) have dramatically improved cancer survival, these drugs cause hypertension in a majority of patients. This side effect is often dose limiting and increases cardiovascular mortality in cancer survivors. This review summarizes recent advances in our understanding of the molecular mechanisms and clinical findings that impact management of VEGFRi-induced hypertension. Recent studies define new connections between endothelial dysfunction and VEGFRi-induced hypertension, including the balance between nitric oxide, oxidative stress, endothelin signaling, and prostaglandins and the potential role of microparticles, vascular smooth muscle cells, vascular stiffness, and microvessel rarefaction. Data implicating genetic polymorphisms that might identify patients at risk for VEGFRi-induced hypertension and the growing body of literature associating VEGFRi-induced hypertension with antitumor efficacy are reviewed. These recent advances have implications for the future of cardio-oncology clinics and the management of VEGFRi-induced hypertension.

New Insights into Profibrotic Myofibroblast Formation in Systemic Sclerosis: When the Vascular Wall Becomes the Enemy.

In systemic sclerosis (SSc), abnormalities in microvessel morphology occur early and evolve into a distinctive vasculopathy that relentlessly advances in parallel with the development of tissue fibrosis orchestrated by myofibroblasts in nearly all affected organs. Our knowledge of the cellular and molecular mechanisms underlying such a unique relationship between SSc-related vasculopathy and fibrosis has profoundly changed over the last few years. Indeed, increasing evidence has suggested that endothelial-to-mesenchymal transition (EndoMT), a process in which profibrotic myofibroblasts originate from endothelial cells, may take center stage in SSc pathogenesis. While in arterioles and small arteries EndoMT may lead to the accumulation of myofibroblasts within the vessel wall and development of fibroproliferative vascular lesions, in capillary vessels it may instead result in vascular destruction and formation of myofibroblasts that migrate into the perivascular space with consequent tissue fibrosis and microvessel rarefaction, which are hallmarks of SSc. Besides endothelial cells, other vascular wall-resident cells, such as pericytes and vascular smooth muscle cells, may acquire a myofibroblast-like synthetic phenotype contributing to both SSc-related vascular dysfunction and fibrosis. A deeper understanding of the mechanisms underlying the differentiation of myofibroblasts inside the vessel wall provides the rationale for novel targeted therapeutic strategies for the treatment of SSc.

Blood-brain barrier leakage and perivascular collagen accumulation precede microvessel rarefaction and memory impairment in a chronic hypertension animal model.

Hypertension (HT) is one of the main causes of vascular dementia, lead to cognitive decline. Here, we investigated the relationship between cerebral microvessels, pericytes, extracellular matrix (ECM) accumulation, blood-brain barrier (BBB) breakdown, and memory impairment at mid-life in a chronic hypertension animal model. Spontaneously hypertensive rats (SHRs) (n = 20) are chosen for the model and age matched Wistar rats (n = 16) as controls. Changes in brain microvasculature and in vitro experiments are shown with immunofluorescence studies and cognition with open field, novel object recognition, and Y maze tests. There was a significant reduction in pericyte coverage in SHRs (p = 0.021), while the quantitative parameters of the cerebral microvascular network were not different between groups. On the other hand, parenchymal albumin leakage, as a Blood-brain barrier (BBB) breakdown marker, was prominent in SHRs (p = 0.023). Extracellular matrix (ECM) components, collagen type 1, 3 and 4 were significantly increased (accumulated) around microvasculature in SHRs (p = 0.011, p = 0.013, p = 0.037, respectively). Furthermore, in vitro experiments demonstrated that human brain vascular pericytes but not astrocytes and endothelial cells secreted type I collagen upon TGFβ1 exposure pointing out a possible role of pericytes in increased collagen accumulation around cerebral microvasculature due to HT. Furthermore, valsartan treatment decreased the amount of collagen type 1 secreted by pericytes after TGFβ1 exposure. At the time of evaluation, SHRs did not demonstrate cognitive decline and memory impairments. Our results showed that chronic HT causes ECM accumulation and BBB leakage before leading to memory impairments and therefore, pericytes could be a novel target for preventing vascular dementia.

Exercise Recovers Loss of Microvessel Density due to Lowered Nitric Oxide Bioavailability

Microvessel rarefaction is an indicator of a challenged or disease state in both humans and animal models. The loss of peripheral microvessel density is associated with the metabolic syndrome (MetS). Decreased microvessel density significantly increases the risk for vascular dysfunction and peripheral vascular disease (PVD). Nitric oxide (NO) has been shown to have pro‐angiogenic properties, which suggests that improvement of NO bioavailability in MetS can augment microvessel rarefaction. Exercise, when used as an intervention during MetS, can improve NO bioavailability. The initiating factors for microvessel rarefaction still remain unclear and whether or not exercise can reverse the rarefaction associated with MetS needs to be determined. To examine the correlation between loss in microvessel density and decrease in nitric oxide (NO) bioavailability, capillary to fiber ratio was measured in the soleus and gracilis muscles of obese Zucker rats (OZRs), a model of MetS, and in lean healthy Zucker rats (LZRs). NO levels were measured in stimulated aortas of both groups. OZRs and LZRs were separated into sedentary and exercise training groups (consisting of treadmill running 1 hr/d, 5 d/wk for 8 weeks at 70% of their max running speed). The gracilis arteriole was isolated and then cannulated in a pressurized vessel myobath and exposed to increasing doses of acetylcholine (ACh) to test endothelium‐dependent dilation. Capillary density in the soleus was significantly lower by 15% in OZRs compared to LZRs (p<0.05), and exercise training did not have an effect. In the gracilis, exercise improved capillary density in both OZRs and LZRs. The difference in capillary density between OZR and LZR controls in the gracilis suggest a genotypic disparity. NO bioavailability was significantly decreased in sedentary OZR compared to sedentary LZR, and exercise augmented NO in both groups (p<0.05). Gracilis arteriole dilation response was lower in OZR than LZR (p<0.01) but exercise improved dilation in OZR to almost LZR control values. These results suggest that there is a correlation between decreased NO bioavailability and the loss of peripheral microvessel density, and that exercise may at least partially, depending on the muscle type, reverse this effect and recover arteriole reactivity.Support or Funding InformationThe authors gratefully acknowledge the support by National Institute of Health (5P20GM109098)

Allogenic adipose-derived stem cell therapy overcomes ischemia-induced microvessel rarefaction in the myocardium: systems biology study

BackgroundMyocardial microvascular loss after myocardial infarction (MI) remains a therapeutic challenge. Autologous stem cell therapy was considered as an alternative; however, it has shown modest benefits due to the impairing effects of cardiovascular risk factors on stem cells. Allogenic adipose-derived stem cells (ASCs) may overcome such limitations, and because of their low immunogenicity and paracrine potential may be good candidates for cell therapy. In the present study we investigated the effects of allogenic ASCs and their released products on cardiac rarefaction post MI.MethodsPig subcutaneous adipose tissue ASCs were isolated, expanded and GFP-labeled. ASC angiogenic function was assessed by the in-vivo chick chorioallantoic membrane (CAM) model. Pigs underwent MI induction and 7 days after were randomized to receive: allogenic ASCs (intracoronary infusion); conditioned media (CM; intravenous infusion); ASCs + CM; or PBS/placebo (control). Cardiac damage and function were monitored by 3-T cardiac magnetic resonance imaging upon infusion (baseline CMR) and 1 and 3 weeks thereafter. We assessed in the myocardium: microvessel density; angiogenic markers (CD105, CD31, TF, VEGFR2, VEGFR1, vWF, eNOS, CD62); collagen deposition; and reparative fibrosis (TGFβ/TβRII/collagen). Differential proteomics of ASCs and CM was performed to characterize the ASC protein signature.ResultsCAM indicated a significant ASC proangiogenic capacity. In pigs after MI, only PBS/placebo animals displayed an impaired cardiac function 3 weeks after infusion (p < 0.05 vs baseline). Administration of ASCs + CM significantly enhanced neovessel formation and favored cardiac repair post MI (p < 0.05 vs the other groups). Molecular markers of angiogenesis were significantly upregulated both at transcriptional and protein levels (p < 0.05). The in-silico bioinformatics analysis of the ASC and CM proteome (interactome) indicated activation of a coordinated protein network involved in the formation of microvessels and the resolution of rarefaction.ConclusionCoadministration of allogenic ASCs and their CM synergistically contribute to the neovascularization of the infarcted myocardium through a coordinated upregulation of the proangiogenic protein interactome.

The NRF2 knockout rat: a new animal model to study endothelial dysfunction, oxidant stress, and microvascular rarefaction.

Nuclear factor (erythroid-derived 2)-like-2 (NRF2) is a master antioxidant and cell protective transcription factor that upregulates antioxidant defenses. In this study we developed a strain of Nrf2 null mutant rats to evaluate the role of reduced NRF2-regulated antioxidant defenses in contributing to endothelial dysfunction and impaired angiogenic responses during salt-induced ANG II suppression. Nrf2(-/-) mutant rats were developed using transcription activator-like effector nuclease technology in the Sprague-Dawley genetic background, and exhibited a 41-bp deletion that included the start codon for Nrf2 and an absence of immunohistochemically detectable NRF2 protein. Expression of mRNA for the NRF2-regulated indicator enzymes heme oxygenase-1, catalase, superoxide dismutase 1, superoxide dismutase 2, and glutathione reductase was significantly lower in livers of Nrf2(-/-) mutant rats fed high salt (HS; 4% NaCl) for 2 wk compared with wild-type controls. Endothelium-dependent dilation to acetylcholine was similar in isolated middle cerebral arteries (MCA) of Nrf2(-/-) mutant rats and wild-type littermates fed low-salt (0.4% NaCl) diet, and was eliminated by short-term (3 days) HS diet in both strains. Low-dose ANG II infusion (100 ng/kg sc) reversed salt-induced endothelial dysfunction in MCA and prevented microvessel rarefaction in wild-type rats fed HS diet, but not in Nrf2(-/-) mutant rats. The results of this study indicate that suppression of NRF2 antioxidant defenses plays an essential role in the development of salt-induced oxidant stress, endothelial dysfunction, and microvessel rarefaction in normotensive rats and emphasize the potential therapeutic benefits of directly upregulating NRF2-mediated antioxidant defenses to ameliorate vascular oxidant stress in humans.

Evidence for a Profound Remodeling of Skeletal Muscle and Its Microvasculature in Sickle Cell Anemia

Sickle cell anemia (SCA) is a hemoglobinopathy leading to major hematologic, hemorheologic, and hemodynamic disorders that induce various complications, including organ failure, and ultimately lead to death. Here, we assessed for the first time repercussions of SCA on skeletal muscle and its microvasculature. Twenty-seven sedentary Cameroonian volunteer men participated in the study. They were assigned to one of three groups according to their hemoglobin status (healthy control subjects, n = 10; sickle cell trait carriers, n = 10; and SCA patients, n = 7) and underwent muscle biopsy of the vastus lateralis. SCA was associated with microvessel rarefaction, decrease in capillary tortuosity, and widening of microvessel diameter. The absence of capillary wall reinforcement was shown by lack of wall thickening and lack of fibrous tissue or smooth muscle in their constitution. We also observed changes in fiber type distribution, muscle atrophy, an increase in satellite cell number, and a decrease in activity of creatine kinase and several oxidative enzymes. No signs of tissue necrosis, inflammatory stress, fibrosis, or segmented fibers were observed. The present study highlighted marked effects of SCA on microvascular, structural, and energetic characteristics of skeletal muscle.

High VEGF Serum Levels on Days +50 and +100 after Allogeneic Stem Cell Transplantation Predict Severe Chronic GvHD

IntroductionSevere chronic graft versus host disease (cGvHD) is one of the main complications following allogeneic stem cell transplantation (SCT) and a major contributor to late treatment-related morbidity and mortality. The symptoms of the condition mimic autoimmune diseases like scleroderma, Sjögren syndrome, primary biliary cirrhosis, wasting syndrome or bronchiolitis obliterans.The pathogenesis of GVHD is still poorly understood. However, increasing evidence suggests that endothelial injury and angiogenesis are involved. Chronic GvHD associates with a rarefaction of microvessels in the affected tissue. (Tichelli A. et al. 2008).We therefore hypothesized that VEGF serum levels could be used to predict occurrence of chronic GvHD.MethodsWritten informed consent to sample and data collection in accordance with the declaration of Helsinki was obtained from 394 patients undergoing SCT between 2002-2011 at our institution. Blood serum samples were obtained on day 0, day 50 and day 100 following transplantation. Concentrations of VEGF were quantified in patient sera by the multiplex protein array technology (Luminex) according to the manufacturer’s instructions for protein multiplexing (Bio-Rad).The occurrence of mild and severe cGvHD was evaluated retrospectively by chart review using the NIH Consensus criteria in chronic Graft versus Host Disease (Filipovic et al., 2005). A score of 3 in the clinical scoring of organ systems suggested in the consensus criteria was considered severe cGvHD. In cGvHD of the lung a score of 2 and higher was considered severe. Isolated elevations of bilirubin or liver enzymes were only considered cGvHD if confirmed by histopathology. Time to onset of mild and/or severe cGvHD and organ systems involved were recorded. All statistical calculations were performed using SPSS19. The rates of mild and severe cGvHD were plotted using cumulative incidence analysis of cause-specific hazards and compared in various groups using log rank test.ResultsThe median age of patients was 52 years (17 - 70). 242 patients (61%) were male and 152 (39%) were female. Patients were suffering from a broad range of underlying diseases (SAA 5, ALL 42, AML 119, Amyloidosis 1, CLL 29, other B-NHL 52, T-NHL 12, CML 15, other MPS 19, MDS 32, HD 5, MM 61, sarcoma 2). 148 (38%) patients had a matched family donor (MFD), 156 (40%) had a matched unrelated donor (MUD), 90 (23%) had a mismatched unrelated donor (MMUD). 308 (78%) patients received myeloablative conditioning (MAC), 86 (22%) received reduced intensity conditioning (RIC). 281 (71%) patients received ATG, 113 (29%) did not receive ATG. 166 patients (42%) developed mild cGvHD and 75 (19%) developed severe cGvHD. 24 (6%) patients developed sclerodermatous changes or fasciitis, 17 (4%) developed severe lung cGvHD and 26 (7%) developed severe gastrointestinal GvHD. The median time of onset of severe cGvHD was 11.18 months (1.4- 88.9).Median serum concentrations of VEGF on days +50 and +100 in those patients developing severe cGvHD were markedly elevated as compared to those patients developing no or only mild cGvHD: i) d+50: no cGvHD 119.5 (4.3-1577.5) pg/ml, mild cGvHD 113,8 (9.1-620.7) pg/ml, severe cGvHD 158.11 (22.5-415.3) pg/ml; p=0.044). ii) d+100: no cGvHD 107.8 (7.8-753.3) pg/l, mild cGvHD 95.4 (15.5-561.9) pg/ml, severe cGvHD 158.1 (20.2-607.2) pg/ml, p=0.048). Moreover, VEGF levels on days +50 and +100 in those patients developing severe lung GvHD, sclerodermatous lesions or severe gastrointestinal GvHD were found to be elevated (Figure 1). High serum concentrations of VEGF did not correlate with acute GvHD of any grade or steroid refractory acute GvHD. Serum concentrations of VEGF >150 pg/ml on day +100 after allogeneic stem cell transplantation were associated with a 2.1 fold higher rate of severe cGvHD (p=0.001).ConclusionsThese results suggest that VEGF serum levels on day +50 and day +100 after allogeneic stem cell transplantation may be useful for early prediction of severe cGvHD. One explanation for prognostic VEGF elevations occurring long before onset of clinical cGVHD could be that endothelial cell alterations are involved in the pathogenesis of severe cGvHD which develop early but become only relevant after tapering immunosuppression [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Temporally distributed mechanisms for skeletal muscle microvascular rarefaction in metabolic syndrome (674.9)

Evolution of metabolic syndrome (MetSyn) and its constituent pathologies is associated with a gradual reduction in skeletal muscle microvessel density (MVD), known as rarefaction. While contributing to elevated vascular resistance at high metabolic demand and impairing mass transport/exchange, the temporal development of rarefaction and the contributing mechanisms which lead to the progressive microvessel loss are critical areas for investigation. Our studies demonstrate that overall reductions to MVD in skeletal muscle of obese Zucker rats (OZR) are well predicted by a chronic loss in vascular nitric oxide (NO) bioavailability. However, prior to any loss in NO, skeletal muscle arterioles from OZR exhibit an early shift in ROS, inflammatory biomarkers and arachidonic acid metabolism, resulting in increased production of TxA2. This increases venular leukocyte adhesion and causes a second, localized oxidant pulse, which initiates microvascular degradation in post‐capillary venules that is strongly associated with plasma MCP‐1 levels. If NO bioavailability is maintained (TEMPOL) after the initial increase in TxA2 and microvessel loss, the total rarefaction magnitude is blunted. Alternately, if actions of TxA2 are chronically inhibited, the initial rarefaction never develops and the subsequent secondary pulse (from NO reductions) is strongly muted. A similar muting of the initial and secondary pulse of rarefaction is manifested if TNF‐α production is inhibited (pentoxifyllene). These results suggest that microvessel rarefaction in OZR represents the integration of an early mild vessel loss that is dependent on TxA2, which creates an environment for the extensive rarefaction that is dependent on low NO bioavailability.Grant Funding Source: Supported by National Institutes of Health and American Heart Association

Impaired skeletal muscle fatigue resistance in metabolic syndrome is associated with alterations in microvascular hematocrit levels and variability (675.3)

Vascular dysfunction in metabolic syndrome is associated with poor performance of in situ skeletal muscle. Prior to impaired function in skeletal muscle itself, this reflects a failure to effectively perfuse working muscle. While altered vascular reactivity, arteriolar wall stiffness and microvessel rarefaction have been implicated, the accumulated impact on mass transport/exchange is unknown. Using tracer washout and existing models for microvascular hematocrit (HMV) and permeability‐surface area product (PS), we determined these parameters for in situ skeletal muscle of lean (LZR) and obese Zucker (OZR) rats. HMV in OZR was less than for LZR; treatment with adrenergic blockers or anti‐oxidants improved HMV slightly, but imposition of all agents restored HMV fully. PS, reduced in OZR vs. LZR, was not impacted by intervention. Using in situ cremaster muscle, HMV was determined in individual microvessels. Similar to above, HMV was reduced in OZR vs. LZR, although variability was higher; with post‐intervention changes mirroring that from washout experiments. These data suggest a low, spatially variable, HMV and global loss in surface area are contributors to poor muscle performance in OZR.Grant Funding Source: Supported by National Institutes of Health and American Heart Association

Blood Vessel Adaptation with Fluctuations in Capillary Flow Distribution

Throughout the life of animals and human beings, blood vessel systems are continuously adapting their structures – the diameter of vessel lumina, the thickness of vessel walls, and the number of micro-vessels – to meet the changing metabolic demand of the tissue. The competition between an ever decreasing tendency of luminal diameters and an increasing stimulus from the wall shear stress plays a key role in the adaptation of luminal diameters. However, it has been shown in previous studies that the adaptation dynamics based only on these two effects is unstable. In this work, we propose a minimal adaptation model of vessel luminal diameters, in which we take into account the effects of metabolic flow regulation in addition to wall shear stresses and the decreasing tendency of luminal diameters. In particular, we study the role, in the adaptation process, of fluctuations in capillary flow distribution which is an important means of metabolic flow regulation. The fluctuation in the flow of a capillary group is idealized as a switch between two states, i.e., an open-state and a close-state. Using this model, we show that the adaptation of blood vessel system driven by wall shear stress can be efficiently stabilized when the open time ratio responds sensitively to capillary flows. As micro-vessel rarefaction is observed in our simulations with a uniformly decreased open time ratio of capillary flows, our results point to a possible origin of micro-vessel rarefaction, which is believed to induce hypertension.

Pharmacological Inhibition of Microsomal Prostaglandin E Synthase-1 Suppresses Epidermal Growth Factor Receptor-Mediated Tumor Growth and Angiogenesis

BackgroundBlockade of Prostaglandin (PG) E2 production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE2 promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway.Methodology/Principal FindingsHere we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE2 production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE2 production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice.ConclusionOur work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE2 mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.

Increased Vascular Generation of Thromboxane A2: an Initiating Condition for Microvascular Rarefaction in Obese Zucker Rats?

Our studies have demonstrated that rarefaction of the skeletal muscle microcirculation in OZR with evolution of the metabolic syndrome is well predicted by a chronic reduction to vascular nitric oxide (NO) bioavailability. However, prior to any loss in NO, skeletal muscle vessels from OZR exhibit a shift in arachidonic acid metabolism, with an increased production of TxA2 over PGI2. Temporal analyses indicate that this early increase in vascular TxA2 production/action is associated with an initial early rarefaction of 5–7%. If NO bioavailability is maintained (TEMPOL) after the initial increase in TxA2 and microvessel loss, the total rarefaction magnitude is blunted. Alternately, if actions of TxA2 are chronically inhibited, the initial rarefaction never develops and the subsequent secondary pulse (from NO reductions) is strongly muted. Chronic treatment with pentoxyfillene (to prevent TNF‐α production) or HMG CoA reductase inhibitors (atorvastatin/simvastatin) prevented much of the secondary pulse of rarefaction (associated with maintenance of NO bioavailability), yet did not alter either the shift in TxA2 production or the initial rarefaction. These results suggest that microvessel rarefaction in OZR represents the integration of an early mild vessel loss that is dependent on TxA2, which creates an environment for the extensive rarefaction that is dependent on low NO bioavailability. (NIH DK64668)

Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome

The obese Zucker rat (OZR) model of the metabolic syndrome is partly characterized by moderate hypercholesterolemia, in addition to other contributing comorbidities. Previous results suggest that vascular dysfunction in OZR is associated with chronic reduction in vascular nitric-oxide (NO) bioavailability and chronic inflammation, both frequently associated with hypercholesterolemia. As such, we evaluated the impact of chronic cholesterol-reducing therapy on the development of impaired skeletal muscle arteriolar reactivity and microvessel density in OZR and its impact on chronic inflammation and NO bioavailability. Beginning at seven weeks of age, male OZR were treated with gemfibrozil, probucol, atorvastatin, or simvastatin (in chow) for 10 weeks. Subsequently, plasma and vascular samples were collected for biochemical/molecular analyses, while arteriolar reactivity and microvessel network structure were assessed by using established methodologies after 3, 6, and 10 weeks of drug therapy. All interventions were equally effective at reducing total cholesterol, although only the statins also blunted the progressive reductions to vascular NO bioavailability, evidenced by greater maintenance of acetylcholine-induced dilator responses, an attenuation of adrenergic constrictor reactivity, and an improvement in agonist-induced NO production. Comparably, while minimal improvements to arteriolar wall mechanics were identified with any of the interventions, chronic statin treatment reduced the rate of microvessel rarefaction in OZR. Associated with these improvements was a striking statin-induced reduction in inflammation in OZR, such that numerous markers of inflammation were correlated with improved microvascular reactivity and density. However, using multivariate discriminant analyses, plasma RANTES (regulated on activation, normal T-cell expressed and secreted), interleukin-10, monocyte chemoattractant protein-1, and tumor necrosis factor alpha were determined to be the strongest contributors to differences between groups, although their relative importance varied with time. While the positive impact of chronic statin treatment on vascular outcomes in the metabolic syndrome are independent of changes to total cholesterol, and are more strongly associated with improvements to vascular NO bioavailability and attenuated inflammation, these results provide both a spatial and temporal framework for targeted investigation into mechanistic determinants of vasculopathy in the metabolic syndrome.

Angiogenesis in the kidney: a new therapeutic target?

The prevalence of chronic kidney disease has been growing consistently for the past decades. Renal failure is often associated with defective angiogenesis, and recognition of the contribution of the renal microcirculation to the progression of chronic renal disease may aid in the development of therapeutic interventions. Intra-renal proliferation, remodeling, and/or rarefaction of microvessels in response to injury can all aggravate nephron damage, and experimental evidence suggests that they may constitute the early steps in the complex pathways involved in progressive renal injury. Recent studies showed the benefits of targeted interventions deemed to promote neovascularization (e.g. progenitor cells, growth factors) on the ischemic myocardium and brain and in a few models of renal disease. Evidence of aberrant renal microvascular architecture in various forms of renal disease provides the impetus to attempt modulating the renal microcirculation to interfere with the disease process. Targeted interventions to preserve the renal microcirculation may not only decrease the evolving injury in renal vascular disease but also potentially constitute a coadjuvant intervention to become part of a comprehensive management plan to improve the success of parallel strategies to preserve renal function, such as revascularization.

Proteinase-activated receptor-1 mediates allogeneic CD8+ T cell-induced apoptosis of vascular endothelial cells

Vascular endothelial-cells injury plays a pivotal role in the pathogenesis of graft-versus-host disease (GVHD) and transplant-associated endothelial injury syndrome. Vascular endothelial cells are an exposed target tissue for immune-mediated injury during GVHD. Early endothelial injury syndromes share common features with acute GVHD. Chronic GVHD leads to a rarefaction of microvessels caused by the infiltration of alloreactive cytotoxic T lymphocytes. In this context, allogeneic reactive cytotoxic T cell may contribute to apoptosis of vascular endothelial cells. The involvement of proteinase-activated receptor (PAR-1) in regulation of apoptosis has been recently recognized in many cell types. We hypothesized that apoptosis of vascular endothelial cells induced by allogeneic cytotoxic T cell are mediated via the PAR-1. Allogeneic CD8(+) T cell, PAR-1 agonist peptide (SFLLRN) induced apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) as assessed by AnnexinV-FITC labeling. To ascertain the mechanism of endothelial apoptosis, we determined that allogeneic CD8(+) T cell, SFLLRN enhanced cleavage of caspase-3 and led to p38MAPK activation as assessed by Western blot. The effects of allogeneic CD8(+) T cell and SFLLRN on apoptosis of vascular endothelial cells were largely prevented by a cleavage-blocking anti-human PAR-1-antibody (ATAP2) and a specific inhibitor of p38MAPK. In concert, these observations provide strong evidence that allogeneic CD8(+) T cell induces apoptosis of human vascular endothelial cells through PAR-1-dependent modulation of intrinsic apoptotic pathway via alterations of p38MAPK and caspase-3.

Increased myocardial prevalence of C‐reactive protein in human coronary heart disease: relation to local endothelial injury and microvessel rarefaction

Increased plasma C-reactive protein (CRP) is a biomarker of coronary heart disease (CHD) but its potential roles as a disease participant are not well defined. A key component of CHD is endothelial injury and dysfunction. Here we tested the hypotheses that CRP is detectable in human cardiac specimens and is elevated in patients with CHD, and these changes are related to local endothelial stress and/or microvessel rarefaction. Cardiac LV specimens were collected at autopsy within 4h of death and were classified as normal controls (CTL) or evidence of CHD (w/o MI) (n=12/group). Immunohistochemistry and digital image analyses were used to define discrete regional distributions of CRP in myocyte, coronary artery and microvessel regions. Prevalence of subendocardial microvessels was determined using an anti-CD31 antibody and automated macro. Increased interstitial fibrosis & hypertrophy was seen in CHD. CTL had detectable levels of CRP, which was increased in CHD group (p<0.05). Microvessel prevalence was significantly reduced in CHD myocardium vs. CTL, and rarefaction was inversely correlated to myocardial CRP prevalence (p<0.05). These findings provide evidence that locally produced myocardial CRP is a potential participant of myocardial toxicity and microvascular rarefaction. These observations may also help to explain the strong predictive value of increased blood CRP to CHD progression in humans

Conductance and Resistance Vessels in Arterial Hypertension

More than one-fourth of individuals in the Eastern populations are hypertensive. Both large (conductance) arteries and small (resistance) arterioles are involved in the physiopathology of hypertension. Large arteries from hypertensive subjects are stiffer and thicker than in normotensive matched controls. Stiffer arterial walls are responsible for higher pulse wave velocity and earlier reflection wave in hypertensives leads to increased systolic pressure and pulse pressure and finally to increased left ventricular after-load. Evidence for these pathophysiological mechanisms arises from studies of pulsatile arterial hemodynamics, as highlighted recently by the role of PWV and wave reflections as independent factors in cardiovascular risk in hypertension. Resistance arterioles (200-30 μm) are characterized by the presence of a myogenic tone able to protect the capillary bed against abnormally high blood pressure and to control the local tissue blood flow. Several types of alterations of resistance vessels are characteristics of chronic hypertension. -reduced lumen diameter in relation with exaggerated vasoconstriction, - hypertrophy of the vascular wall resulting in decreased lumen size and increased wall-to-lumen ratio, - rarefaction of microvessels i.e. arterioles and capillaries. Experimental and clinical results allowed to evidence that antihypertensive drugs might reverse the structural changes of the large and resistance arteries. Through modification in the timing of wave reflections, it is also possible to reduce the disproportionate increase in systolic blood pressure and the associated cardiovascular risk. Some recent trials are aiming to reduce or reverse microvascular network rarefaction. This could be a promising way not only to normalize arterial blood pressure but also to reduce target organs complications. Keywords: Arterial Stiffness, mean blood pressure (MBP), hypertrophy, hypertensive rats (SHR), Endothelium Dysfunction