Excessive Endothelial Cell Research Articles

Novel mathematical approach to accurately quantify 3D endothelial cell morphology and vessel geometry based on fluorescently marked endothelial cell contours: Application to the dorsal aorta of wild-type and Endoglin-deficient zebrafish embryos.

Endothelial cells, which line the lumen of blood vessels, locally sense and respond to blood flow. In response to altered blood flow dynamics during early embryonic development, these cells undergo shape changes that directly affect vessel geometry: In the dorsal aorta of zebrafish embryos, elongation of endothelial cells in the direction of flow between 48 and 72 hours post fertilization (hpf) reduces the vessel's diameter. This remodeling process requires Endoglin; excessive endothelial cell growth in the protein's absence results in vessel diameter increases. To understand how these changes in vessel geometry emerge from morphological changes of individual endothelial cells, we developed a novel mathematical approach that allows 3D reconstruction and quantification of both dorsal aorta geometry and endothelial cell surface morphology. Based on fluorescently marked endothelial cell contours, we inferred cross-sections of the dorsal aorta that accounted for dorsal flattening of the vessel. By projection of endothelial cell contours onto the estimated cross-sections and subsequent triangulation, we finally reconstructed 3D surfaces of the individual cells. By simultaneously reconstructing vessel cross-sections and cell surfaces, we found in an exploratory analysis that morphology varied between endothelial cells located in different sectors of the dorsal aorta in both wild-type and Endoglin-deficient zebrafish embryos: In wild-types, ventral endothelial cells were smaller and more elongated in flow direction than dorsal endothelial cells at both 48 hpf and 72 hpf. Although dorsal and ventral endothelial cells in Endoglin-deficient embryos had similar sizes at 48 hpf, dorsal endothelial cells were much larger at 72 hpf. In Endoglin-deficient embryos, elongation in flow direction increased between 48 hpf and 72 hpf in ventral endothelial cells but hardly changed in dorsal endothelial cells. Hereby, we provide evidence that dorsal endothelial cells contribute most to the disparate changes in dorsal aorta diameter in wild-type and Endoglin-deficient embryos between 48 hpf and 72 hpf.

The testicular microvasculature in Klinefelter syndrome is immature with compromised integrity and characterized by excessive inflammatory cross-talk.

Does Klinefelter syndrome (KS) lead to a distinct gene expression pattern at single-cell level in the testes that could provide insight into the reported microvascular dysfunction in the testes? A distinct gene expression pattern within microvascular-associated cells of males with KS suggests excessive endothelial cell (EC) activation, disorganized vessel formation, and the presence of immature vessels with compromised integrity. Recent studies show that males with KS exhibit microvascular dysfunction in their testes, which affects blood flow and is associated with lower circulating levels of testosterone. A comparative cross-sectional study of males with KS (n = 6), non-obstructive azoospermia (NOA) (n = 5), cryptozoospermia (n = 3), and controls (n = 15) was carried out. We analyzed publicly available single-cell RNA sequencing data of testicular cells from males with KS, males with NOA, males with cryptozoospermia, and controls. The integration of these datasets allowed us to analyze gene expression profiles and communication patterns among the cell types within the testis and to identify capillary ECs to investigate changes at the microvascular level. Rooted in changes at the single-cell level, our study demonstrates a shift in gene expression forming the foundation for altered cellular communication, microvascular remodeling, and pro-inflammatory responses within the testes of males with KS. We identified genes that were dysregulated in capillary ECs from males with KS (Padj < 0.05). Specifically, the unique microvascular gene expression in males with KS indicated enhanced capillary EC activation and increased inflammatory cross-talk, leading to impaired vessel maturation and increased EC barrier permeability. Our study is constrained by an unbalanced design, with varying sample sizes and number of cells within each group. We acknowledge the restricted access to clinical information. In addition, our findings were deduced from changes in gene expression, which limits us to infer potential biological consequences arising from these alterations. Furthermore, the absence of a pre-pubertal age group limits the generalizability of our findings and warrants further investigation. This study offers novel insights into the testicular pathophysiology in KS and underscores the potential contribution of microvascular dysfunction to the hypogonadism and infertility observed in males with KS. While this study aims to better understand the microvascular dysfunction in KS, the precise connections to testosterone deficiency and testicular atrophy remain to be fully elucidated. A.S. was supported by the Independent Research Fund Denmark (0134-00130B). C.H.G. was supported by Novo Nordisk Foundation (NNF15OC0016474, NNF20OC0060610), 'Fonden til lægevidenskabens fremme', the Familien Hede Nielsen foundation and the Independent Research Fund Denmark (0134-00406A). E.B.J. was supported by Aarhus University and E.B.J. and C.H.G by the Independent Research Fund Denmark (2096-00165A). J.M.K. was supported by Lundbeckfonden (R307-2018-3667), Carlsberg Fonden (CF19-0687), Novo Nordisk Fonden (0073440) and Steno Diabetes Center Aarhus (SDCA). The authors declare no conflicts of interest. N/A.

R-Ras Deficiency in Pericytes Causes Frequent Microphthalmia and Perturbs Retinal Vascular Development

Purpose: The retinal vasculature is heavily invested by pericytes. Small GTPase R-Ras is highly expressed in endothelial cells and pericytes, suggesting importance of this Ras homolog for the regulation of the blood vessel wall. We investigated the specific contribution of pericyte-expressed R-Ras to the development of the retinal vasculature. Methods: The effect of R-Ras deficiency in pericytes was analyzed in pericyte-targeted conditional Rras knockout mice at birth and during the capillary plexus formation in the neonatal retina. Results: The offspring of these mice frequently exhibited unilateral microphthalmia. Analyses of the developing retinal vasculature in the eyes without microphthalmia revealed excessive endothelial cell proliferation, sprouting, and branching of the capillary plexus in these animals. These vessels were structurally defective with diminished pericyte coverage and basement membrane formation. Furthermore, these vessels showed reduced VE-cadherin staining and significantly elevated plasma leakage indicating the breakdown of the blood-retinal barrier. This defect was associated with considerable macrophage infiltration in the retina. Conclusions: The normal retinal vascular development is dependent on R-Ras expression in pericytes, and the absence of it leads to unattenuated angiogenesis and significantly weakens the blood-retinal barrier. Our findings underscore the importance of R-Ras for pericyte function during the normal eye development.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE−/−) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE−/− and Nox2 (ESMIRO/ApoE−/−/Nox2−/y) were generated and compared with ESMIRO/ApoE−/−/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE−/− mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE−/−/Nox2−/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE−/−/Nox2−/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE−/− mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Abstract 097: Interaction of miRNA, Endothelial Mineralocorticoid Receptor and Epithelial Sodium Channel in Endothelium Stiffness and Aortic Dysfunction

Excessive endothelial cell (EC) mineralocorticoid receptor (ECMR) and epithelial sodium channel activation in endothelium (EnNaC) increase oxidative stress and inflammation with associated cardiovascular abnormalities. Previous studies implicate elevations in circulating aldosterone (Aldo) in obesity enhance ECMR/EnNaC signaling that contribute to the development of vascular stiffness, in part, by reducing endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the specific role of ECMR/EnNaC signaling and its molecular mechanisms have not been explored. We hypothesized interactions between ECMR, dysregulation of miRNA expression, and EnNaC contribute to obese-/Aldo-induced endothelium dysfunction and aortic stiffness. Six week-old ECMR -/- and wild type littermate mice were fed a mouse chow or Western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. The EnNaC alpha subunit KO (EnNaC -/- ) and EnNaC +/+ female mice were administrated Aldo (250 μg/kg/day) via osmotic minipumps for 3 weeks beginning at 25-28 weeks of age. RNA sequencing showed that in ECMR +/+ mice WD induced increased miR-7a, miR-34, and miR-6973a and reduced miR-99, miR-486a, miR-6904, miR-6916, miR-6240 that potentially target EnNaC expression. For EnNaC in vascular function, EnNaC -/- significantly reduced inward sodium currents by patch clamp in isolated mouse ECs. Meanwhile, EnNaC -/- significantly inhibited Aldo-induced endothelium stiffness and endothelium dependent relaxation observed in EnNaC +/+ . Further, chronic Aldo infusion induced aortic endoplasmic reticulum stress, reduced eNOS activation, endothelium permeability, expression of pro-inflammatory cytokines IL1 and IL6, oxidative stress, and aortic collagen 1 deposition and these abnormalities were attenuated in EnNaC -/- mice. These data indicate that interaction of endothelial specific ECMR/miRNA/EnNaC mediates vascular endothelium dysfunction and prompts aortic stiffness.

Mechanical Activation of Hypoxia-Inducible Factor 1α Drives Endothelial Dysfunction at Atheroprone Sites.

Atherosclerosis develops near branches and bends of arteries that are exposed to low shear stress (mechanical drag). These sites are characterized by excessive endothelial cell (EC) proliferation and inflammation that promote lesion initiation. The transcription factor HIF1α (hypoxia-inducible factor 1α) is canonically activated by hypoxia and has a role in plaque neovascularization. We studied the influence of shear stress on HIF1α activation and the contribution of this noncanonical pathway to lesion initiation. Quantitative polymerase chain reaction and en face staining revealed that HIF1α was expressed preferentially at low shear stress regions of porcine and murine arteries. Low shear stress induced HIF1α in cultured EC in the presence of atmospheric oxygen. The mechanism involves the transcription factor nuclear factor-κB that induced HIF1α transcripts and induction of the deubiquitinating enzyme Cezanne that stabilized HIF1α protein. Gene silencing revealed that HIF1α enhanced proliferation and inflammatory activation in EC exposed to low shear stress via induction of glycolysis enzymes. We validated this observation by imposing low shear stress in murine carotid arteries (partial ligation) that upregulated the expression of HIF1α, glycolysis enzymes, and inflammatory genes and enhanced EC proliferation. EC-specific genetic deletion of HIF1α in hypercholesterolemic apolipoprotein E-defecient mice reduced inflammation and endothelial proliferation in partially ligated arteries, indicating that HIF1α drives inflammation and vascular dysfunction at low shear stress regions. Mechanical low shear stress activates HIF1α at atheroprone regions of arteries via nuclear factor-κB and Cezanne. HIF1α promotes atherosclerosis initiation at these sites by inducing excessive EC proliferation and inflammation via the induction of glycolysis enzymes.

The mTOR/AP-1/VEGF signaling pathway regulates vascular endothelial cell growth

Vascular restenosis is a common adverse event following percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). The atypical Ser/Thr protein kinase mammalian target of rapamycin (mTOR) plays an important role in cell differentiation and apoptosis. Vascular restenosis caused by excessive endothelial cell proliferation can be inhibited by local application of the mTOR inhibitor rapamycin (RAPA); however, RAPA can also suppress normal vascular endothelial cell growth by blocking mTOR/VEGF signaling, although the underlying mechanism is still unclear. Here, endogenous mTOR, AP-1, and VEGF were inhibited or overexpressed to investigate the mechanism underlying the effects of RAPA. Inhibition of AP-1 or mTOR with AP-1-siRNA or RAPA treatment respectively, decreased vascular endothelial cell proliferation, upregulation of AP-1 or mTOR increased cell proliferation, and VEGF overexpression increased, while RAPA-induced mTOR inhibition decreased vascular endothelial cell proliferation, the results indicate that combining mTOR downregulation and VEGF upregulation might both inhibit restenosis and maintain normal vascular endothelial cell growth after PCI or CABG, suggest the mTOR/AP-1/VEGF pathway might play a crucial role in regulating vascular endothelial cell growth.

To be EndMT or not to be, that is the question in pulmonary hypertension.

To be EndMT or not to be, that is the question in pulmonary hypertension.

Endothelial-Specific Notch Blockade Inhibits Vascular Function and Tumor Growth through an eNOS-Dependent Mechanism

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels.

Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance–induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance–mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance–related vascular disease.

E: TARGETING NOX2 NADPH OXIDASE IN INSULIN RESISTANCE RELATED ENDOTHELIAL DYSFUNCTION

Laboratory based cell culture studies have shown that reactive oxygen species (ROS) induced oxidative stress as an important molecular mechanism in the development and progression of cardiovascular diseases. Despite that, large scale clinical trials have shown that general antioxidants are not only ineffective in treating cardiovascular diseases but also, in some cases, can have harmful effects. In this context, the next logical step is to specifically targeting critical molecules in ROS generation rather than using non-specific antioxidants. Insulin resistance, the antecedent of type 2 diabetes mellitus, can itself initiate various vascular complications of diabetes before the development of clinically diagnosable diabetes. Insulin resistance is characterised by excessive endothelial cell generation of cytotoxic concentrations of ROS. NADPH oxidases (NOX), a group of ROS producing enzymes, have been recently shown as the major source of excessive ROS in insulin resistant endothelial cells. We hypothesised that specific targeting of NOX could be the way forward in developing antioxidant molecules as therapeutic agents for vascular diseases. In this study, we examined the role of NOX enzymes and specifically Nox2 isoform in superoxide generation in 2 complementary in vivo models (endothelial specific and whole body) of human insulin resistance. Using gp91-ds tat peptide, a non-specific NOX inhibitor, we have specifically targeted NOX generated superoxide production. For acute inhibition we have treated cells or tissues with 50 M gp91ds-tat or control peptide for 30 minutes. For chronic inhibition, mice were implanted with osmotic mini-pumps which delivered 10 mg/kg/day of drug for 28 days. The excessive level of superoxide found in insulin resistant endothelial cells was significantly reduced by acute or chronic treatment with gp91ds-tat peptide. Also NO dependent vasorelaxation response, which was impaired in insulin resistance, was improved with gp91ds-tat peptide treatment. Since insulin resistant endothelial cells showed higher expression of Nox2 isoform, Nox2 gene was knocked down using siRNA which significantly reduced superoxide levels. Furthermore, to clearly examine the involvement of Nox2 isoform in this context, double transgenic mice with endothelial specific insulin resistance and Nox2 gene knockout were generated. These mice showed reduced superoxide production and improved vascular function. Thus Nox2 is the critical molecule in insulin resistance induced endothelial dysfunction. In conclusion, our study establishes that pharmacological inhibition of NOX as a novel way to treat insulin resistance related vascular disease.

195 TARGETING NOX2 NADPH OXIDASE IN INSULIN RESISTANCE RELATED ENDOTHELIAL DYSFUNCTION

Laboratory based cell culture studies have shown that reactive oxygen species (ROS) induced oxidative stress as an important molecular mechanism in the development and progression of cardiovascular diseases. Despite that, large scale clinical trials have shown that general antioxidants are not only ineffective in treating cardiovascular diseases but also, in some cases, can have harmful effects. In this context, the next logical step is to specifically targeting critical molecules in ROS generation rather than using non-specific antioxidants. Insulin resistance, the antecedent of type 2 diabetes mellitus, can itself initiate various vascular complications of diabetes before the development of clinically diagnosable diabetes. Insulin resistance is characterised by excessive endothelial cell generation of cytotoxic concentrations of ROS. NADPH oxidases (NOX), a group of ROS producing enzymes, have been recently shown as the major source of excessive ROS in insulin resistant endothelial cells. We hypothesised that specific targeting of NOX could be the way forward in developing antioxidant molecules as therapeutic agents for vascular diseases. In this study, we examined the role of NOX enzymes and specifically Nox2 isoform in superoxide generation in 2 complementary in vivo models (endothelial specific and whole body) of human insulin resistance. Using gp91-ds tat peptide, a non-specific NOX inhibitor, we have specifically targeted NOX generated superoxide production. For acute inhibition we have treated cells or tissues with 50 M gp91ds-tat or control peptide for 30 minutes. For chronic inhibition, mice were implanted with osmotic mini-pumps which delivered 10 mg/kg/day of drug for 28 days. The excessive level of superoxide found in insulin resistant endothelial cells was significantly reduced by acute or chronic treatment with gp91ds-tat peptide. Also NO dependent vasorelaxation response, which was impaired in insulin resistance, was improved with gp91ds-tat peptide treatment. Since insulin resistant endothelial cells showed higher expression of Nox2 isoform, Nox2 gene was knocked down using siRNA which significantly reduced superoxide levels. Furthermore, to clearly examine the involvement of Nox2 isoform in this context, double transgenic mice with endothelial specific insulin resistance and Nox2 gene knockout were generated. These mice showed reduced superoxide production and improved vascular function. Thus Nox2 is the critical molecule in insulin resistance induced endothelial dysfunction. In conclusion, our study establishes that pharmacological inhibition of NOX as a novel way to treat insulin resistance related vascular disease.

Uncoupling of the VEGF-endothelial nitric oxide axis in diabetic nephropathy: an explanation for the paradoxical effects of VEGF in renal disease

In many forms of experimental kidney diseases, renal VEGF is low, and administering VEGF can be shown to be protective. A paradox occurs in diabetes, in which renal VEGF levels are high and a deleterious effect of VEGF on kidney disease has been shown. We have hypothesized that endothelial dysfunction induced by hyperglycemia or other factors may underlie the pathogenic mechanisms of a high VEGF state. VEGF normally stimulates endothelial nitric oxide (NO) release and acts in concert with elevated NO levels as a trophic factor for vascular endothelium. The increased NO derived from the endothelial cell acts as an inhibitory factor that prevents excess endothelial cell proliferation, vascular smooth muscle cell proliferation, and macrophage infiltration. In the setting where NO bioavailability is reduced in diabetes, high levels of VEGF lead to excessive endothelial cell proliferation, stimulation of macrophage chemotaxis, and vascular smooth muscle cell activation. Consistent with this hypothesis is our recent observation that diabetes induced in endothelial NO-deficient mice results in clinical and histological features identical to human diabetic nephropathy. The discovery of the key role for impaired endothelial NO bioavailability in the stimulation of VEGF and VEGF-dependent disease may provide key insights into not only the pathogenesis of diabetic nephropathy but also the utility and hazard of administering VEGF as a treatment for kidney disease.

Histomorphometrical study in cavernous lymphangioma of the tongue

To study the histomorphometrical characteristics of lymphatic vessels in cavernous lymphangiomas of the tongue. Immunohistochemical stainings were prepared in the 20 specimens with three antibodies [D2-40, CD31 and proliferating cell nuclear antigen (PCNA)]. Three-dimensional (3D) reconstruction and histometrical analysis of the lymphatic vessels was also examined. Distinctly positive staining for D2-40 was found in dilated lymphatic vessels located in the lamina propria beneath the thinned covering epithelium. Small blood vessels stained positively for CD31 were present in the lamina propria. PCNA-positive lymphatic endothelial cells were scattered in both control and lymphangioma. The 3D architecture of lymphatic vessels was characterized by a complex network with irregular branches in the lamina propria. Histometrical analysis showed that the number of lymphatic endothelial cells per lymphatic vessel perimeter in cavernous lymphangioma was significantly higher than that in control. There were no significant differences in the lymphatic density and the ratio of PCNA-positive lymphatic endothelial cell nuclei to the total number of lymphatic endothelial cell nuclei between control and lymphangioma. These results indicate the absence of excessive endothelial cell proliferation in dilated lymphatic vessels in cavernous lymphangioma. Cavernous lymphangioma may be attributed to the enlargement of lymphatic vessels without the tumorous proliferation of lymphatic endothelial cells.

Matrix Metalloproteinases in the Urine of Patients With Pulmonary Arterial Hypertension

Associated pulmonary arterial hypertension (APAH) is a disease of vascular remodeling of the lung involving excessive endothelial and smooth-muscle cell proliferation. We hypothesized that matrix metalloproteinases (MMPs) would be frequently detected in the urine of patients with APAH and that MMP species would correlate with disease etiology. We studied a cohort of 48 patients with APAH and 47 age- and sex-matched control subjects. We performed substrate gel electrophoresis supported by immunoblot analysis using monospecific antibodies on urine samples to detect the presence of MMPs. We collected clinical and hemodynamic data and correlated these variables to the urinary MMP pattern. We studied 35 patients with primary idiopathic pulmonary hypertension (iPAH) and 13 patients with other forms of pulmonary hypertension (APAH-other). Detection of at least one MMP species was significantly higher in the APAH group (75%) compared to normal control subjects (17%, p < 0.001). The detection of MMPs in the urine was highest in iPAH (80%). Four MMP species were highly detected in patients with APAH compared to control subjects: > 150-kd species, 140-kd species, 92-kd species (MMP-9), and 72-kd species (MMP-2). Significant differences in MMP species were observed between the iPAH and APAH-other groups, with MMP-2 observed in 60% of patients with iPAH but only 15% of APAH-other patients (p < 0.01). Urinary MMPs are detected in a significantly higher proportion of patients with APAH compared to control subjects and are likely to reflect the pulmonary vascular remodeling process. Urinary MMP species show correlation with disease etiology subtype.

Endoluminal aortic stent grafts — morphology of complications

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745–752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.

Increased Tie2 Expression, Enhanced Response to Angiopoietin-1, and Dysregulated Angiopoietin-2 Expression in Hemangioma-Derived Endothelial Cells

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745-752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.

Lipoteichoic Acid inhibits Lipopolysaccharide-Induced Adhesion Molecule Expression and IL-8 Release in Human Lung Microvascular Endothelial Cells

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-alpha, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3-30 microg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 microg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-alpha on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 microg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to approximately 2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.

Therapeutic Angiogenesis in Ischemic Heart Disease

Introduction: Coronary heart disease is the leading cause of death in the Western world. Traditionally, patients with coronary heart disease requiring a revascularizing procedure have had to undergo either coronary artery bypass surgery, which usually involves open thoracotomy, or percutaneous transluminal balloon angioplasty, or a related procedure. Unfortunately, especially in patients with severe diffuse coronary heart disease, revascularization by these means can be difficult or even impossible. It has been shown that incomplete revascularization is a predictor of a worsened postoperative outcome (i.e., recurrent angina, myocardial infarction, or even death). We have known for a long time that many patients with ischemic disease develop angiographically visible collateral vessels. Initial research focused on the enlargement of preexisting collateral vessels, a process for which Wolfgang Schaper has proposed the term “neoarteriogenesis.”1 It is now clear that true “angiogenesis,” defined as the formation of new vessels by sprouting from preexisting vessels, also occurs. The latter also should be differentiated from the earliest embryological process of new vessel formation directly from mesodermal endothelial cell precursors, which is called “vasculogenesis.”2 In the last few years, clinical trials have been initiated with the goal of enhancing angiogenesis to treat peripheral vascular disease and ischemic heart disease. In this review, we will summarize the underlying concepts for this novel mode of therapy, plus the available information on its efficacy. The sprouting of capillaries from preexisting vessels (angiogenesis) is a normal and necessary process to supply the growing organism with nutrients and oxygen. Ischemic vascular disease, along with wound healing and the monthly endometrial proliferation, are conditions in which angiogenesis may be beneficial or necessary. Of course, angiogenesis is also part of the pathogenic mechanism of tumors, hemangiomas, proliferative retinopathies, and inflammatory diseases like rheumatoid arthritis and psoriasis.3 Even in ischemic vascular disease, it is possible that vascularization of atherosclerotic plaques by vessels arising from the vasa vasorum leads to hemorrhages with consequent plaque instability,4 and thus enhancing angiogenesis may promote vessel pathology. The vasculature of a 70 kg adult is lined by approximately 1,000 m2 of quiescent endothelial cells with a very low turnover rate that can exceed 1,000 days.3 Upon angiogenic activation (e.g., by growth factors released during ischemia), a local inflammatory reaction often can be observed with increased local vascular permeability, vasodilation, and accumulation of monocytes and macrophages. These latter cells release more cytokines and growth factors, which lead to the accumulation of additional inflammatory cells. They also release enzymes that promote the proteolytic degradation of the underlying extracellular matrix and basal membrane. Such degradation causes the endothelial cells to detach from their neighboring cells and the underlying matrix, followed by chemotactic migration and proliferation. Subsequently, formation of a lumen occurs and, eventually, maturation and growth of the newly formed vessel.2,5,6 In vessels larger than capillaries, vascular smooth muscle cells proliferate and migrate as well. In addition, it recently has been shown that circulating endothelial cell precursors released from the bone marrow participate in the formation of new vessels in the setting of tissue ischemia.7 Investigators currently are assessing the importance and contribution of the latter mechanism to neovascularization in the adult organism. If it proves substantial, the traditional means of differentiating between angiogenesis and vasculogenesis may need to be reconsidered.

Excessive Endothelial Cell Proliferation Occurring in an Organizing Thyroid Hematoma: Report of a Case and Review of the Literature.

The authors report a case of a thyroid hematoma containing an exuberant endothelial cell proliferation. No underlying vascular neoplasm was apparent and the hematoma occurred in a substernal goiter from a 90-yr-old male. Review of the literature revealed four similar previously reported cases of endothelial cell proliferation found in thyroid hematomas for a total of five cases. A fine needle aspiration or biopsy procedure antedated the definitive surgery in four patients and may be responsible for the formation of the hematoma in these cases. Areas of the endothelial proliferation resembled papillary endothelial hyperplasia (Masson's hemangioma) in four cases and cavernous hemangioma in three. However, these lesions occurred outside of vascular lumina and instead were intimately associated with fibrinous material of the hematoma. In addition, two cases contained foci of endothelial cells with nuclear atypia or intracytoplasmic lumens requiring distinction from angiosarcoma. These vigorous endothelial proliferations most likely represent alterations in the organization of a thyroid hematoma, a process similar to papillary endothelial hyperplasia, which is usually intravascular. Primary vascular neoplasms of the thyroid are exceedingly rare. Nevertheless, awareness of exuberant endothelial cell proliferation as a potential, albeit rare, manifestation of a thyroid hematoma should prompt careful attention to distinguish this reactive process from vascular neoplasms such as hemangioma or angiosarcoma.