Human Vascular Disease Research Articles

A novel model of persistent retinal neovascularization for the development of sustained anti-VEGF therapies

Anti-vascular endothelial growth factor (VEGF) therapies lead to a major breakthrough in treatment of neovascular retinal diseases such as age-related macular degeneration or diabetic retinopathy. Current management of these conditions require regular and frequent intravitreal injections to prevent disease recurrence once the effect of the injected drug wears off. This has led to a pressing clinical need of developing sustained release formulations or therapies with longer duration. A major drawback in developing such therapies is that the currently available animal models show spontaneous regression of vascular leakage. They therefore not only fail to recapitulate retinal vascular disease in humans, but also prevent to discern if regression is due to prolonged therapeutic effect or simply reflects spontaneous healing.Here, we described the development of a novel rabbit model of persistent retinal neovascularization (PRNV). Retinal Müller glial are essential for maintaining the integrity of the blood-retinal barrier. Intravitreal injection of DL-alpha-aminoadipic acid (DL-AAA), a selective retinal glial (Müller) cell toxin, results in persistent vascular leakage for up to 48 weeks. We demonstrated that VEGF concentrations were significantly increased in vitreous suggesting VEGF plays a significant role in mediating the leakage observed. Intravitreal administration of anti-VEGF drugs (e.g. bevacizumab, ranibizumab and aflibercept) suppresses vascular leakage for 8–10 weeks, before recurrence of leakage to pre-treatment levels. All three anti-VEGF drugs are very effective in re-ducing angiographic leakage in PRNV model, and aflibercept demonstrated a longer duration of action compared with the others, reminiscent of what is observed with these drugs in human in the clinical setting. Therefore, this model provides a unique tool to evaluate novel anti-VEGF formulations and therapies with respect to their duration of action in comparison to the currently used drugs.

<em>In Vivo</em> Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

ENHANCED LYSYL OXIDASE EXPRESSION IN PERIPHERAL ARTERY RESTENOSIS IS ASSOCIATED WITH ALTERED COLLAGENS: IMPLICATIONS IN RESTENOTIC PLAQUE PROGRESSION AND THERAPEUTIC INTERVENTION

Lysyl oxidase (LOX) is an enzyme expressed by vascular smooth muscle cells (smc) and fibroblasts, that maintains extracellular matrix stability and arterial remodeling. Previously we have reported human peripheral vascular disease restenosis showing increased smc and fibroblast proliferation.

AGE-RELATED MORPHOMETRIC CHARACTERISTICS OF REMODELING OF ARTERIAL BED OF HIND LIMBS IN WHITE RATS WITH EXPERIMENTAL HYPERCHOLESTEROLEMIA

Background. The human organism is constantly affected by metabolic risk factors. All of them for a long time are asymptomatic and often form in early childhood long before clinical manifestations. However, morphogenesis and morphofunctional features of age-related structural changes in blood vessels of different sites are poorly studied.Objective. To evaluate the morphometric characteristics of arterial bed remodeling of hind limbs in white rats of PRA and RA with experimentally induced hypercholesterolemia.Methods. The tissues biopsies from femoral, knee and shin areas were studied by means of histological and morphometric methods.Results. Experimental hypercholesterolemia caused remodeling of vessels with increasing thickness of medial and endothelial layer of vessels in the investigated parts of the rats’ body. In this case, the external diameter of vessels increased only in femoral area in the reproductive group of rats. In the group of pre-productive animals, the external diameter of the vessels studied was more or less unaffected. Thus, increased thickness of intima and media led to a decrease in arteries capacity. The depth of vascular lesions and the decrease in functional capacity of blood vessels were directly proportional to the duration of the experiment. In the group of reproductive rats, the changes in vascular bed were more evident.Conclusions. Objective study of the processes allows providing a scientific basis for further research and understanding of the pathomorphism of vascular diseases in humans.

Abstract 123: Perivascular Adiponectin Attenuates the Neointimal Response Potentially via Modulation of Hydrogen Sulfide

Objective: Intimal hyperplasia (IH) limits the durability of vascular interventions. Perivascular adipose serves as scaffolding for vessels and also as a source of adipokines, including adiponectin (APN). The globular domain of adiponectin (gAPN) has high affinity for endothelial AdipoR1 receptors and holds beneficial roles in vascular adaptations. Hydrogen sulfide (H 2 S) is a vasculo-protective gasotransmitter produced by cystathionine γ-lyase (CGL), and low circulating H 2 S levels associate with human vascular disease. We therefore hypothesized that APN knockout (KO) mice would be predisposed to IH, and that local administration of gAPN would protect APN KO mice from IH. Furthermore, we evaluated potential links between APN and H 2 S in these effects. Approach and Results: Wildtype (WT, n=7) and APN KO (n=8) mice maintained on high fat diet were subject to carotid focal stenosis. An additional group of APN KO mice (n=7) were treated by local injection of gAPN suspended in Matrigel. Carotids were harvested 28 days following surgery and analyzed 400-2800μm proximal to the stenosis microscopically. IH area was significantly increased at 400μm in APN KO mice compared to WT (p=<0.05, Fig. A), while gAPN significantly attenuated intimal area compared to untreated KO mice (p=<0.05, Fig. B). This finding was coupled with significant (p=<0.05, Fig. C) decrease in pre-operative baseline serum H 2 S production capacity in APN KO (lead acetate assay) compared to WT. Serum collected upon harvest trended toward increased H 2 S production capacity in APN KO mice treated with gAPN (Fig. D). Kidney CGL expression (Western) revealed a trend for increased in CGL expression in the gAPN group (Fig. E). Conclusion: In a focal stenosis model APN KO mice demonstrated increased distal IH; when administered locally, gAPN significantly attenuated IH. These data also uncover a reduction in H 2 S production capacity in APN KO mice, providing a novel potential link between these potent vasomediators.

Polymorphisms in the selectin gene cluster are associated with fertility and survival time in a population of Holstein Friesian cows.

Selectins are adhesion molecules, which mediate attachment between leucocytes and endothelium. They aid extravasation of leucocytes from blood into inflamed tissue during the mammary gland’s response to infection. Selectins are also involved in attachment of the conceptus to the endometrium and subsequent placental development. Poor fertility and udder health are major causes for culling dairy cows. The three identified bovine selectin genes SELP, SELL and SELE are located in a gene cluster. SELP is the most polymorphic of these genes. Several SNP in SELP and SELE are associated with human vascular disease, while SELP SNP rs6127 has been associated with recurrent pregnancy loss in women. This study describes the results of a gene association study for SNP in SELP (n = 5), SELL (n = 2) and SELE (n = 1) with fertility, milk production and longevity traits in a population of 337 Holstein Friesian dairy cows. Blood samples for PCR-RFLP were collected at 6 months of age and animals were monitored until either culling or 2,340 days from birth. Three SNP in SELPEx4-6 formed a haplotype block containing a Glu/Ala substitution at rs42312260. This region was associated with poor fertility and reduced survival times. SELPEx8 (rs378218397) coded for a Val475Met variant locus in the linking region between consensus repeats 4 and 5, which may influence glycosylation. The synonymous SNP rs110045112 in SELEEx14 deviated from Hardy Weinberg equilibrium. For both this SNP and rs378218397 there were too few AA homozygotes present in the population and AG heterozygotes had significantly worse fertility than GG homozygotes. Small changes in milk production associated with some SNP could not account for the reduced fertility and only SELPEx6 showed any association with somatic cell count. These results suggest that polymorphisms in SELP and SELE are associated with the likelihood of successful pregnancy, potentially through compromised implantation and placental development.

Echocardiographic Pulmonary Artery Systolic Pressure in the Coronary Artery Risk Development in Young Adults (CARDIA) Study: Associations With Race and Metabolic Dysregulation.

BackgroundThe determinants of pulmonary artery systolic pressure (PASP) are not fully understood. It is unknown whether racial differences in PASP exist or if other population characteristics are associated with pulmonary pressure in humans. We examined echocardiographically estimated PASP in the Coronary Artery Risk Development in Young Adults (CARDIA) study, a middle‐aged, biracial community‐based cohort.Methods and ResultsAt the CARDIA year‐25 examination, 3469 participants underwent echocardiography, including measurement of tricuspid regurgitant jet velocity to estimate PASP. Clinical features, laboratory values, pulmonary function tests, and measurement of adipose depot volume were analyzed for association with PASP. PASP was estimated in 1311 individuals (61% female, 51% white). Older age, higher blood pressure, and higher body mass index were associated with higher PASP. Black race was associated with higher PASP after adjustment for demographics and left and right ventricular function (β 0.94, 95% CI 0.24‐1.64; P=0.009), but this association was no longer significant after further adjustment for lung volume (β 0.42, 95% CI −0.68 to 0.96; P=0.74). Insulin resistance, inflammation (C‐reactive protein and interleukin‐6), and visceral adipose volume were independently associated with higher PASP after adjustment for relevant covariates. PASP rose with worsening diastolic function (ratio of early transmitral Doppler velocity to average mitral annular tissue Doppler velocity [E/e′] and left atrial volume index).ConclusionsIn a large biracial cohort of middle‐aged adults, we identified associations among black race, insulin resistance, and diastolic dysfunction with higher echocardiographically estimated PASP. Further studies are needed to examine racial differences in PASP and whether insulin resistance directly contributes to pulmonary vascular disease in humans.

Intravascular hemolysis and the pathophysiology of sickle cell disease.

Hemolysis is a fundamental feature of sickle cell anemia that contributes to its pathophysiology and phenotypic variability. Decompartmentalized hemoglobin, arginase 1, asymmetric dimethylarginine, and adenine nucleotides are all products of hemolysis that promote vasomotor dysfunction, proliferative vasculopathy, and a multitude of clinical complications of pulmonary and systemic vasculopathy, including pulmonary hypertension, leg ulcers, priapism, chronic kidney disease, and large-artery ischemic stroke. Nitric oxide (NO) is inactivated by cell-free hemoglobin in a dioxygenation reaction that also oxidizes hemoglobin to methemoglobin, a non-oxygen-binding form of hemoglobin that readily loses heme. Circulating hemoglobin and heme represent erythrocytic danger-associated molecular pattern (eDAMP) molecules, which activate the innate immune system and endothelium to an inflammatory, proadhesive state that promotes sickle vaso-occlusion and acute lung injury in murine models of sickle cell disease. Intravascular hemolysis can impair NO bioavailability and cause oxidative stress, altering redox balance and amplifying physiological processes that govern blood flow, hemostasis, inflammation, and angiogenesis. These pathological responses promote regional vasoconstriction and subsequent blood vessel remodeling. Thus, intravascular hemolysis represents an intrinsic mechanism for human vascular disease that manifests clinical complications in sickle cell disease and other chronic hereditary or acquired hemolytic anemias.

Human cytomegalovirus infection and vascular disease risk: A meta-analysis

BackgroundHuman cytomegalovirus (HCMV) infection has been associated with the acceleration of vascular disease. Numbers studies were conducted to analyze the association between HCMV infection and risk of vascular disease, but no clear consensus had been reached. The aim of this study was to confirm this relationship precisely by doing a systematic review and meta-analysis. MethodsWe identified relevant studies through a search of PubMed and Embase. Studies were eligible for inclusion if they fulfilled all of the following selection criteria: (1) evaluating the association between HCMV infection and vascular disease; (2) case-control studies or nested case-control studies; (3) and supply the numbers (or percentage) of positivity for HCMV infection in cases and controls, respectively. Data were extracted and analyzed independently by two investigators. Ultimately, We included data from 68 studies, which altogether enrolled 12027 cases and 15386 controls from 24 countries. ResultsHCMV IgG was detected 7376 in 10611 cases, HCMV IgM was detected 153 in 1486 cases and HCMV DNA was detected 654 in 2139 cases. Overall, people exposed to HCMV infection had higher risk than those not exposed for vascular disease (OR 1.70 [95% CI 1.43–2.03] IgG-based HCMV tests, 2.88 [95% CI 1.87–4.43] IgM-based HCMV tests and 2.56 [95% CI 1.46–4.49 PCR-based HCMV tests]). HCMV infection was clearly identified as a risk factor for vascular disease in Asian group, Caucasian group and other group, especially Asian group(OR 1.86 [95% CI 1.33–2.60] IgG-based HCMV tests, 3.57 [95% CI 1.94–6.60] IgM-based HCMV tests and 4.09 [95% CI 3.10–5.40 PCR-based HCMV tests]). ConclusionThis meta-analysis suggested that HCMV infection is associated with an increased risk for vascular disease.

Testosterone-dependent sex differences in red blood cell hemolysis in storage, stress, and disease.

Red blood cell (RBC) hemolysis represents an intrinsic mechanism for human vascular disease. Intravascular hemolysis releases hemoglobin and other metabolites that inhibit nitric oxide signaling and drive oxidative and inflammatory stress. Although these pathways are important in disease pathogenesis, genetic and population modifiers of hemolysis, including sex, have not been established. We studied sex differences in storage or stress-induced hemolysis in RBC units from the United States and Canada in 22 inbred mouse strains and in patients with sickle cell disease (SCD) using measures of hemolysis in 315 patients who had homozygous SS hemoglobin from the Walk-PHASST cohort. A mouse model also was used to evaluate posttransfusion recovery of stored RBCs, and gonadectomy was used to determine the mechanisms related to sex hormones. An analysis of predisposition to hemolysis based on sex revealed that male RBCs consistently exhibit increased susceptibility to hemolysis compared with females in response to routine cold storage, under osmotic or oxidative stress, after transfusion in mice, and in patients with SCD. The sex difference is intrinsic to the RBC and is not mediated by plasmatic factors or female sex hormones. Importantly, orchiectomy in mice improves RBC storage stability and posttransfusion recovery, whereas testosterone repletion therapy exacerbates hemolytic response to osmotic or oxidative stress. Our findings suggest that testosterone increases susceptibility to hemolysis across human diseases, suggesting that male sex may modulate clinical outcomes in blood storage and SCD and establishing a role for donor genetic variables in the viability of stored RBCs and in human hemolytic diseases.

Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions.

Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined, growth factor- and serum-free system by temporal modulation of Wnt/β-catenin signaling via small molecules. We demonstrate a 10-day, two-stage process that recapitulates endothelial cell development, in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described.

Egr-1 identifies neointimal remodeling and relates to progression in human pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is hallmarked by the development of neointimal lesions. The transcription factor Egr-1 seems to play a critical role in neointimal formation in experimental PAH and was identified as a putative target for intervention. In this study we investigated whether Egr-1 is also associated with neointimal-type vascular remodeling in different forms of human PAH or pulmonary hypertension. Using immunohistochemistry, we studied Egr-1 expression specifically in a wide morphologic spectrum of pulmonary arteries in the lung tissue of 72 patients with different forms and stages of PAH, specifically idiopathic PAH (n = 18), advanced-stage congenital heart disease‒associated PAH (PAH-CHD) (n = 21), early-stage PAH-CHD (n = 19) and non-neointimal hypoxic pulmonary hypertension (PH) (n = 4), and controls (n = 10). In PAH patients, pulmonary vascular expression of Egr-1 protein was abundant, whereas it was sporadic in non-neointimal (hypoxic) PH patients and controls. In PAH-CHD, protein expression was more pronounced in patients with advanced vascular lesions compared to those with less advanced lesions, such as medial hypertrophy. Pulmonary vascular Egr-1 expression is significantly increased in patients with PAH, appears specifically associated with neointimal-type vascular remodeling, and correlates with disease progression. These data translate the critical role of Egr-1 in the development of experimental PAH to human pulmonary vascular disease forms.

Phosphodiesterase 1 regulation is a key mechanism in vascular aging.

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; β=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; β=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy.

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice.

Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4% w/w lard) or a high-fat [HF; 21% w/w lard and cholesterol (0/15% w/w)] diet with different B vitamin compositions for 16weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P<0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P<0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis.

Human validation of genes associated with a murine atherosclerotic phenotype

Background: The genetically modified mouse is the most widely applied animal model for studying the pathogenesis of atherosclerotic disease. However, the validity for extrapolation to human vascular occlusive disease is ambiguous. In this study we assessed cardiovascular traits in human GWAS studies of genes identified in atherosclerotic murine models.

Abstract 15954: Both Tetrahydrobiopterin Depletion and eNOS S-Glutathionytion Contribute to eNOS Uncoupling in Coronary Disease Patients

Background: Vascular endothelial dysfunction (VED) is associated with increased oxidative stress due to eNOS uncoupling with increased superoxide (•O2-) production and has important prognostic clinical implications for subsequent cardiovascular events. Our recent studies in hypertensive rats revealed a novel redox-regulated pathway through which eNOS is uncoupled due to S-glutathionylation. However, its role in human vascular disease remains unclear. Hence, we studied eNOS S-glutathionylation and uncoupling and in aortic tissue from patients undergoing cardiovascular surgery and heart transplantation (N=25). Methods: Studies were performed using vessels from spontaneously hypertensive rats (SHRs), control WKY rats and aortic tissue from patients undergoing cardiovascular surgery. S-glutathionylation was determined with anti-glutathione and anti-eNOS, and an antibody specific for eNOS S-glutathionylation at C689 (anti-eNOS Cys689-SG). •O2- was measured using fluorescent probe DHE in the absence/presence of the NOS inhibitor, L-NAME, DTT, or the SOD mimetic, MnTBAP. The NOS cofactor, BH4, was measured by HPLC. Results &amp; Conclusion: S-glutathionylation of eNOS (eNOS-SG) was evident by immunohistology in SHR vessels compared to respective WKY control rat vessels. Similarly, immune assays of the aortic samples from vascular disease patients (VD) showed an increase in eNOS-SG protein when compared to no-disease control tissues (ND) and increased •O2- production was detected. Addition of DTT or β-ME decreased the levels of eNOS-SG with decreased •O2- generation. BH4 content was markedly decreased in the vessels of patients with coronary disease. BH4:BH2 ratio was decreased by over 5-fold also contributing to eNOS uncoupling. This provides the first evidence for an association of both eNOS S-glutathionylation and BH4 depletion leading to eNOS uncoupling with loss of endothelial dependent vasodilation in vessels of coronary disease patients.

Links between ectopic fat and vascular disease in humans.

The average of overweight individual can have differential fat depots in target organs or specific compartments of the body. This ectopic fat distribution may be more of a predictive factor for cardiovascular risk than obesity. Abdominal visceral obesity, a representative ectopic fat, is robustly associated with insulin resistance and cardiovascular risk. Fat depots in the liver and muscle tissue cause adverse cardiometabolic risk by affecting glucose and lipid metabolism. Pericardial fat and perivascular fat affect coronary atherosclerosis, cardiac function, and hemodynamics. Fat around the neck is associated with systemic vascular resistance. Fat around the kidney may increase blood pressure and induce albuminuria. Fat accumulation in or around the pancreas alters glucose metabolism, conferring cardiovascular risk. Ectopic fat may act as an active endocrine and paracrine organ that releases various bioactive mediators that influence insulin resistance, glucose and lipid metabolism, coagulation, and inflammation, which all contribute to cardiovascular risk. Because both obese and apparently lean individuals can have ectopic fat, regional fat distribution may play an important role in the development of cardiovascular diseases in both nonobese and obese people.

Recapitulating physiological and pathological shear stress and oxygen to model vasculature in health and disease.

Studying human vascular disease in conventional cell cultures and in animal models does not effectively mimic the complex vascular microenvironment and may not accurately predict vascular responses in humans. We utilized a microfluidic device to recapitulate both shear stress and O2 levels in health and disease, establishing a microfluidic vascular model (μVM). Maintaining human endothelial cells (ECs) in healthy-mimicking conditions resulted in conversion to a physiological phenotype namely cell elongation, reduced proliferation, lowered angiogenic gene expression and formation of actin cortical rim and continuous barrier. We next examined the responses of the healthy μVM to a vasotoxic cancer drug, 5-Fluorouracil (5-FU), in comparison with an in vivo mouse model. We found that 5-FU does not induce apoptosis rather vascular hyperpermeability, which can be alleviated by Resveratrol treatment. This effect was confirmed by in vivo findings identifying a vasoprotecting strategy by the adjunct therapy of 5-FU with Resveratrol. The μVM of ischemic disease demonstrated the transition of ECs from a quiescent to an activated state, with higher proliferation rate, upregulation of angiogenic genes, and impaired barrier integrity. The μVM offers opportunities to study and predict human ECs with physiologically relevant phenotypes in healthy, pathological and drug-treated environments.

Abstract 522: Shotgun Proteomics Implicates Matrix Proteolysis, Cell Adhesion and Cell Death in Urokinase-Accelerated Vascular Disease

Introduction: Elevated activity of urokinase plasminogen activator (uPA) in human arteries associates with atherosclerosis, aneurysms, and plaque rupture. However, the underlying mechanisms remain unknown. ApoE null mice with macrophage-specific uPA overexpression (SR-uPA mice) have elevated vascular uPA activity, accelerated atherosclerosis, dilated aortas, and histologic features of plaque rupture. Thus, SR-uPA mice may be a useful model of uPA-accelerated human vascular disease. Hypothesis: We hypothesized that mechanisms of uPA-accelerated vascular disease would be revealed by shotgun proteomics of aortas of SR-uPA and nontransgenic (ntg) ApoE null mice. We focused our studies on extracellular matrix (ECM) because previous studies implicate uPA in alteration of matrix-associated proteins. Methods: Using aortas, we developed a method for generating tissue extracts (TE) that selectively isolate ECM proteins. We extracted proteins from aortic arches of SR-uPA and ntg ApoE null mice. We also collected conditioned medium (CM) from their explanted aortas. Results: Mass spectrometry-based proteomics demonstrated that aortic TE were greatly enriched in ECM proteins as determined by Gene Ontology (GO) analysis. These TE contained all major and several minor vascular collagens, and many proteoglycans. CM contained a similar number of ECM proteins as TE; however, TE were enriched in collagens, proteoglycans, elastin, and laminins. Using stringent dual statistical criteria and a FDR of 5%, 107 of 1125 proteins were differentially present in SR-uPA aorta. MMP-2, -3, and -10 were increased; laminins, collagen I, fibronectin, and elastin were decreased. CM analysis revealed 1161 proteins; 57 were differentially present in SR-uPA. NuMa1, nuclear lamins, histones, actin, and myosins were increased; PAI-1, Plg, and A2m were decreased. Unbiased GO analysis helped identify proteolysis, ECM, cell adhesion, and cell death as processes that differed significantly between SR-uPA and ntg aortas. Conclusions: Elevated aortic uPA activity alters abundance of a limited number of proteins and affects specific biological processes, implicating ECM proteolysis, altered cell adhesion, and cell death in the pathogenesis of uPA-mediated vascular disease.

Morphogen-induced Platelet Activation and Cell Signalling

Morphogens are signalling molecules that act directly on cells to produce specific cellular responses. This term was originally put forward by Turing (1952) who also described its chemical basis. Most of these secreted proteins signal between the cells. Wolpert (1969) first reported a role of morphogen in cell signalling. The roles of several morphogens (e.g., Transforming Growth Factor- (TGF- ), Hedgehog/ Sonic Hedgehog, Wingless (Wnt) signalling, Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Retinoic Acid (RA) in signal transduction have been studied by scientists in several cell lineages. Assoian et al. (1983) identified major storage sites of TGF- in human platelets. Xie et al. (2003) showed expression of TGF- regulated gene in a mouse mammary gland epithelial cell line. Iwasaki et al. (2000) reported that EGF stimulates growth of vascular smooth muscles that may be involved in genesis of human vascular disease. RA acts as a ligand and diffusible morphogen, the distribution and levels of which are well controlled in embryonic tissues by its regulated synthesis and breakdown. The occurrence of RA signalling in