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Biomedical subjects

William C Sessa

Publications and source records attributed to William C Sessa.

At least 19 recordsLinked to original sources
PubMed2007

The phosphodiesterase 5 inhibitor sildenafil stimulates angiogenesis through a protein kinase G/MAPK pathway.

cGMP-degrading pathways have received little attention in the context of angiogenesis. In the present study we set out to determine whether cGMP-specific phosphodiesterase 5 (PDE5) inhibition affects new blood vessel growth. Incubation of chicken chorioallantoic membranes (CAMs) in vivo with sildenafil increased vascular length in a dose-dependent manner. Moreover, incubation of cultured endothelial cells (ECs) with the PDE5 inhibitor promoted proliferation, migration, and organization into tube-like structures. The effects of sildenafil on the angiogenesis-related properties of EC could be blocked by pre-treatment with the soluble guanylyl cyclase (sGC) inhibitor ODQ or the protein kinase G (PKG) I inhibitor DT-3. In addition, over-expression of sGC in EC led to an enhanced growth and migratory response to sildenafil. To study the signaling pathways implicated in the sildenafil-stimulated angiogenic responses we determined the phosphorylation status of mitogen-activated protein kinase (MAPK) members. Incubation of cells with sildenafil increased both extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation in a time-dependent manner. Inhibition of MEK by PD98059 and p38 with SB203580 blocked sildenafil-induced proliferation and migration, respectively, suggesting that these MAPK members are downstream of PDE5 and mediate the angiogenic effects of sildenafil. PDE5 inhibitors could, thus, be used in disease states where neo-vessel growth is desired.

Animals↗
PubMed2006

Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking.

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.

Animals↗
PubMed2006

Vascular endothelium summary statement I: Health promotion and chronic disease prevention.

The endothelium can be considered a discrete organ with pathophysiologic implications and as such has both diagnostic and therapeutic possibilities. It is essential for the normal function of the cardiovascular, cerebrovascular, renovascular, and pulmonary vascular system. The endothelium is directly involved in the development and progression of heart disease, stroke, peripheral vascular disease, venous thrombosis, insulin resistance, diabetes, chronic kidney failure, tumor growth, metastases and adverse reproductive outcomes for both the mother and her newborn child. Consequently the endothelium represents an objective biological determinant on which to base new multidisciplinary prevention and health promotion strategies. This summary statement suggests some possible avenues for clinical and public health research.

Animals↗
PubMed2006

Evidence supporting changes in Nogo-B levels as a marker of neointimal expansion but not adaptive arterial remodeling.

Both neointimal hyperplasia and inward remodeling contribute to restenosis and lumen loss. Nogo-B has been recently described as an inhibitor of vascular injury and neointimal hyperplasia. To determine whether Nogo-B expression may be a mediator of inward remodeling, we examine the localization of expression of Nogo-B in an in vivo model that examines both neointimal hyperplasia and inward remodeling. The rabbit carotid artery was subjected to balloon injury, outflow branch ligation to reduce flow, or both balloon injury and reduction in flow. In balloon injury-induced neointimal hyperplasia Nogo-B expression was reduced in the intima and media but stimulated in the adventitia. In low flow-induced inward remodeling medial Nogo-B expression was not reduced and adventitial Nogo-B expression was not stimulated. Low flow significantly augmented balloon injury-induced neointimal hyperplasia and was accompanied by reduced intimal and medial Nogo-B expression, and increased adventitial Nogo-B expression in both smooth muscle cells and macrophages. Low flow-induced inward remodeling is not associated with changes in medial Nogo-B expression and is distinct from injury-induced neointimal hyperplasia. Pharmacological strategies to inhibit neointimal hyperplasia and restenosis using normal flow models may only partially account for lumen loss and therefore may not accurately predict responses in patients with extensive outflow disease.

Animals↗
PubMed2006

Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis.

Bmx/Etk non-receptor tyrosine protein kinase has been implicated in endothelial cell migration and tube formation in vitro. However, the role of Bmx in vivo is not known. Bmx is highly induced in the vasculature of ischemic hind limbs. We used both mice with a genetic deletion of Bmx (Bmx-KO mice) and transgenic mice expressing a constitutively active form of Bmx under the endothelial Tie-2 enhancer/promoter (Bmx-SK-Tg mice) to study the role of Bmx in ischemia-mediated arteriogenesis/angiogenesis. In response to ischemia, Bmx-KO mice had markedly reduced, whereas Bmx-SK-Tg mice had enhanced, clinical recovery, limb perfusion, and ischemic reserve capacity when compared with nontransgenic control mice. The functional outcomes in these mice were correlated with ischemia-initiated arteriogenesis, capillary formation, and vessel maturation as well as Bmx-dependent expression/activation of TNF receptor 2- and VEGFR2-mediated (TNFR2/VEGFR2-mediated) angiogenic signaling in both hind limb and bone marrow. More importantly, results of bone marrow transplantation studies showed that Bmx in bone marrow-derived cells plays a critical role in the early phase of ischemic tissue remodeling. Our study provides the first demonstration to our knowledge that Bmx in endothelium and bone marrow plays a critical role in arteriogenesis/angiogenesis in vivo and suggests that Bmx may be a novel target for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.

Animals↗
PubMed2006

Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase.

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

Acylation↗
PubMed2006

Identification of a receptor necessary for Nogo-B stimulated chemotaxis and morphogenesis of endothelial cells.

Nogo isoforms (Nogo-A and -B) have been implicated in regulating neural and cardiovascular functions, such as cell spreading and chemotaxis. Unlike the loop domain (Nogo-66) found in all Nogo isoforms that can interact with a neural-specific Nogo-66 receptor, the receptor for the amino terminus of Nogo-B that mediates vascular function is unknown. Here, we identify a previously uncharacterized Nogo-B receptor specific for the amino terminus of Nogo-B and show that Nogo-B receptor localizes with the ligand Nogo-B during VEGF and wound healing angiogenesis in vivo, mediates chemotaxis in a heterologous expression system and chemotaxis, and 3D tube formation in native endothelial cells. Thus, identification of this receptor may lead to the discovery of agonists or antagonists of this pathway to regulate vascular remodeling and angiogenesis.

Amino Acid Sequence↗
PubMed2006

Essential role of nitric oxide in VEGF-induced, asthma-like angiogenic, inflammatory, mucus, and physiologic responses in the lung.

VEGF, nitric oxide (NO), inflammation, and vascular- and extravascular remodeling coexist in asthma and other disorders. In these responses, VEGF regulates angiogenesis. VEGF also induces inflammation and remodeling. The mechanisms of the latter responses have not been defined, however. We hypothesized that VEGF-induces extravascular tissue responses via NO-dependent mechanisms. To evaluate this hypothesis, we compared the effects of transgenic VEGF165 in lungs from normal mice, mice treated with pan-NO synthase (NOS) or endothelial NOS (eNOS) inhibitors, and mice with null mutations of inducible NOS (iNOS) or eNOS. These studies demonstrate that VEGF selectively stimulates eNOS and iNOS. They also demonstrate that VEGF induces pulmonary alterations via NO-dependent and -independent mechanisms with angiogenesis, edema, mucus metaplasia, airway hyperresponsiveness, lymphocyte accumulation, dendritic cell hyperplasia and S-nitrosoglutathione reductase stimulation being NO-dependent and dendritic cell activation being NO-independent. Furthermore, they demonstrate that eNOS and iNOS both contribute to these responses. NO/NOS-based interventions may be therapeutic in VEGF-driven inflammation and remodeling.

Alcohol Dehydrogenase↗
PubMed2006

Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo.

Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS-/-) mice and their wild-type control as experimental animals, platelet-activating factor (PAF) at 10(-7) m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 +/- 2.2 to a peak net value of 84.4 +/- 2.7 units, while the corresponding values in cremaster muscle of eNOS-/- mice were 1.0 +/- 0.3 and 15.6 +/- 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS-/- mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10(-7) m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation.

Animals↗
PubMed2006

Functional relevance of Golgi- and plasma membrane-localized endothelial NO synthase in reconstituted endothelial cells.

OBJECTIVE: We have previously shown in COS-7 cells that targeting of endothelial NO synthase (eNOS) to the Golgi or plasma membrane (PM) regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (ECs). The goal of the current study was to isolate these 2 pools of enzyme in ECs and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels. METHODS AND RESULTS: Using an RNA interference strategy, we generated stable populations of ECs that had >90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS "knockdown" ECs with Golgi- and PM-targeted eNOS restored the ability of ECs to produce NO. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in ECs. In contrast, Golgi eNOS was less responsive to both calcium- and Akt-dependent agonists. eNOS restricted to the PM was more sensitive to manipulation of membrane cholesterol levels and was significantly attenuated by modified low-density lipoprotein. CONCLUSIONS: Within ECs, the PM is the most efficient location to produce NO but is more vulnerable to cholesterol levels and modified low-density lipoprotein.

Animals↗
PubMed2006

An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice.

Advances in understanding the relationship between protein structure and DNA binding specificity have made it possible to engineer zinc finger protein (ZFP) transcription factors to specifically activate or repress virtually any gene. To evaluate the potential clinical utility of this approach for peripheral vascular disease, we investigated the ability of an engineered vascular endothelial growth factor (VEGFa)-activating ZFP (MVZ+426b) to induce angiogenesis and rescue hindlimb ischemia in a murine model. Hindlimb ischemia was surgically induced in advanced-age C57/BL6 mice. Adenovirus (Ad) encoding either MVZ+426b or the fluorescent marker dsRed was delivered to the adducter muscle of the ischemic hindlimb, and the effects on blood flow, limb salvage, and vascularization were assessed. Ad-MVZ+426b induced expression of VEGFa at the mRNA and protein levels and stimulated a significant increase in vessel counts in the ischemic limb. This was accompanied by significantly increased blood flow and limb salvage as measured serially for 4 wk. These data demonstrate that activation of the endogenous VEGFa gene by an engineered ZFP can induce angiogenesis in a clinically relevant model and further document the feasibility of designing ZFPs to therapeutically regulate gene expression in vivo.

Adenoviridae↗
PubMed2006

Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.

Animals↗
PubMed2006

Mild increases in portal pressure upregulate vascular endothelial growth factor and endothelial nitric oxide synthase in the intestinal microcirculatory bed, leading to a hyperdynamic state.

Increased nitric oxide (NO) is the main factor leading to the hyperdynamic circulation associated with advanced portal hypertension (PHT), but the initial mechanisms and the magnitude of increase in portal pressure required to trigger NO production are not known. We addressed these issues by studying systemic and splanchnic hemodynamics and endothelial NO synthase (eNOS) and VEGF expression in rats with different degrees of portal hypertension. Portal vein ligation (PVL) performed over needles of three different calibers (16-, 18-, and 20-gauge) yielded different degrees of PHT and portosystemic shunting. Compared with sham rats, all three groups of PVL rats exhibited features of hyperdynamic circulation. Rats with minimal portal hypertension (PVL with a 16-gauge needle) showed an early increase in VEGF and eNOS expression selectively at the jejunum. Immunofluorescence showed that VEGF expression was located in highly vascularized areas of the mucosa. Inhibition of VEGF signaling markedly attenuated the increase in eNOS expression. In conclusion, mild increases in portal pressure are enough to upregulate eNOS at the intestinal microcirculation, and this occurs, at least in part, through VEGF upregulation.

Angiogenesis Inhibitors↗
PubMed2006

Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels.

Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared long- and short-term flow-mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow-mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav-1 into the endothelium. In conclusion, these results showed that endothelial Cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels.

Animals↗
PubMed2006

Differential functions of tumor necrosis factor receptor 1 and 2 signaling in ischemia-mediated arteriogenesis and angiogenesis.

We have previously shown that tumor necrosis factor (TNF) acts via its two receptors TNFR1 and TNFR2 to elicit distinct signaling pathways in vascular endothelial cells (ECs). Here we used a femoral artery ligation model to demonstrate that TNFR1-knockout (KO) mice had enhanced, whereas TNFR2-KO had reduced, capacity in clinical recovery, limb perfusion, and ischemic reserve capacity compared with the wild-type mice. Consistently, ischemia-initiated collateral growth (arteriogenesis) in the upper limb and capillary formation and vessel maturation (angiogenesis) in the lower limb were enhanced in TNFR1-KO but were reduced in TNFR2-KO mice. Furthermore, our results suggest that vascular proliferation, but not infiltration of macrophages and lymphocytes, accounted for the phenotypic differences between the TNFR1-KO and TNFR2-KO mice. In wild-type animals TNFR2 protein in vascular endothelium was highly up-regulated in response to ischemia, leading to increased TNFR2-specific signaling as determined by the formation TNFR2-TRAF2 complex and activation of TNFR2-specific kinase Bmx/Etk. In isolated murine ECs, activation of TNFR2 induced nuclear factor-kappaB-dependent reporter gene expression, EC survival, and migration. In contrast, activation of TNFR1 caused inhibition of EC migration and EC apoptosis. These data demonstrate that TNFR1 and TNFR2 play differential roles in ischemia-mediated arteriogenesis and angiogenesis, partly because of their opposite effects on EC survival and migration.

Amino Acid Sequence↗
PubMed2005

PKCalpha activates eNOS and increases arterial blood flow in vivo.

Endothelial nitric oxide synthase (eNOS) plays an important role in control of vascular tone and angiogenesis among other functions. Its regulation is complex and has not been fully established. Several studies have emphasized the importance of phosphorylation in the regulation of eNOS activity. Although it is commonly accepted that protein kinase C (PKC) signaling inhibits eNOS activity by phosphorylating Thr497 and dephosphorylating Ser1179, the distinct role of different PKC isoforms has not been studied so far. The PKC family comprises roughly 12 different isozymes that activate distinct downstream pathways. The present study was designed to investigate the role of PKCalpha isoform in regulation of eNOS activity. Overexpression of PKCalpha in primary endothelial cells was associated with increased eNOS-Ser1179 phosphorylation and increased NO production. Inhibition of PKCalpha activity either by siRNA transfection or by overexpression of a dominant negative mutant resulted in a marked decrease in FGF2-induced Ser1179 phosphorylation and NO production. In vivo, PKCalpha transduction in rat femoral arteries resulted in a significant increase in the resting blood flow that was suppressed by treatment with L-NAME, an eNOS inhibitor. In conclusion, these data demonstrate for the first time that PKCalpha stimulates NO production in endothelial cells and plays a role in regulation of blood flow in vivo.

Animals↗
PubMed2005

Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve.

The genetic loss of endothelial-derived nitric oxide synthase (eNOS) in mice impairs vascular endothelial growth factor (VEGF) and ischemia-initiated blood flow recovery resulting in critical limb ischemia. This result may occur through impaired arteriogenesis, angiogenesis, or mobilization of stem and progenitor cells. Here, we show that after ischemic challenge, eNOS knockout mice [eNOS (-/-)] have defects in arteriogenesis and functional blood flow reserve after muscle stimulation and pericyte recruitment, but no impairment in endothelial progenitor cell recruitment. More importantly, the defects in blood flow recovery, clinical manifestations of ischemia, ischemic reserve capacity, and pericyte recruitment into the growing neovasculature can be rescued by local intramuscular delivery of an adenovirus encoding a constitutively active allele of eNOS, eNOS S1179D, but not a control virus. Collectively, our data suggest that endogenous eNOS-derived NO exerts direct effects in preserving blood flow, thereby promoting arteriogenesis, angiogenesis, and mural cell recruitment to immature angiogenic sprouts.

Animals↗