Irradiation modulates association of NF-Y with histone-modifying cofactors PCAF and HDAC.

Post-irradiation complications including thrombus formation result from increased procoagulant activity of vascular endothelial cells and elevated levels of von Willebrand factor (VWF) contribute to this process. We have previously demonstrated that irradiation induction of the VWF is mediated through interaction of NF-Y transcription factor with its cognate binding site in the VWF promoter. We have also demonstrated that irradiation increases the association of NF-Y with histone acetyltransferase p300/CBP-associated factor (PCAF). We now report that irradiation decreases the association of NF-Y with histone deacetylase 1 (HDAC1). We demonstrate that irradiation-induced changes in association of NF-Y with HDAC1 and PCAF lead to increased PCAF recruitment to the VWF promoter, increased association of acetylated histone H4 with the VWF promoter and subsequently increased transcription. We also demonstrate that this process is correlated to dephosphorylation of HDAC1 and is inhibited by calyculin A, an inhibitor of protein phosphatase1.

Accurate placement of microvascular guide sutures.

A simple technique to accurately place initial microvascular guide sutures is described. Enhanced suture placement enables anastomosis construction in situations in which the clamp must be placed very close to the vessel end or if the vessel lumen is distorted or poorly visualized. Because this technique eliminates the need for retraction on the guide suture tails, it is particularly useful for solo microvascular surgeons.

Superoxide regulation of endothelin-converting enzyme.

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.

Gene therapy in the vasculature and the kidney.

Gene therapy has many potential applications for the treatment of vascular disease. Though a number of tough problems remain to be solved, the potential specificity with which an almost limitless number of mechanisms could be targeted, and the success that has been achieved in animal models in vivo make it likely that we will see further rapid expansion of this technology, and therapeutic use of gene therapy in humans in the future.

Neutralizing TGF-beta1 antibody infusion in neonatal rat delays in vivo glomerular capillary formation 1.

BACKGROUND: The interruption of transforming growth factor-beta (TGF-beta) signaling with dominant negative type II TGF-beta receptors in bovine glomerular endothelial cells abrogates capillary morphogenesis in vitro, and genetic defects in the TGF-beta1 signaling cascade in mice and humans result in abnormalities of blood vessel morphology. This study sought to determine whether TGF-beta1 participates in renal glomerular capillary development in vivo. METHODS: To inhibit TGF-beta1 action, neutralizing anti-TGF-beta1 IgG was infused intra-arterially into the suprarenal aorta of three-day-old rats, and the glomerular endothelial cell appearance was evaluated two days later by immunohistochemical detection of the endothelium-specific von Willebrand factor, in situ analysis of vascular endothelial growth factor receptor binding, and morphometric study of developing glomerular structures by transmission electron microscopy. RESULTS: The infusion of neutralizing the TGF-beta1 antibody markedly reduced the invasion of comma- and S-shaped bodies by endothelial cells, and inhibited organization of endothelial cells into capillaries in these structures. In addition, capillary lumen formation and endothelial cell fenestration in developing cortical, but not in deep, already mature glomeruli were inhibited by neutralizing TGF-beta1 antibody. Seven days after TGF-beta1 antibody infusion, glomeruli appeared normal, and no reduction in glomerular number was observed. CONCLUSIONS: These findings suggest that TGF-beta1 plays a critical role in the formation of glomerular capillaries during renal development in the rat, and that flattening and fenestration of glomerular capillaries require the action of TGF-beta1.

Expression of SET is modulated as a function of cell proliferation.

We explored a biological role of SET as it relates to cell proliferation and differentiation. Immunohistochemical staining demonstrated that the expression of SET was ubiquitous and diffuse over the whole embryo on gestational day 15. At a later stage of development, SET was expressed at relatively lower levels and localized to specific tissues and cells. On embryonic day 19, specific SET immunoreactivity was found in the epithelium of skin, respiratory tract, intestine, and retina as well as in muscle and cartilage. In these cells SET was stained mostly in the nucleus, which was supported indirectly by nuclear transport of enhanced green fluorescence protein-SET fusion proteins in ECV304 endothelial cells. Set mRNA expression was further confirmed in various cultured cells, including NIH 3T3 cells, L6 myoblast cells, human umbilical vein endothelial cells, and ECV304 cells. Using F9 teratocarcinoma cell lines, which were stimulated to differentiate into the two different cell lineages of parietal and visceral endoderm, we have further examined the role of SET. The expression of set mRNA and SET protein was diminished about three-fold in both differentiated endoderm cells compared to the undifferentiated F9 cells. However, when F9 cells were subjected to serum starvation, reduction of set mRNA abundance also took place at a similar level to that observed in response to differentiation. Consistent with this, quiescent L6 myoblast showed a marked downregulation of set mRNA compared to proliferating cells. These results suggest that SET is involved mainly in the regulation of cell proliferation rather than differentiation during embryonic development.

Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness.

OBJECTIVE: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. METHODS: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm2 shear stress and then for an additional 3 days with 1 or 25 dyne/cm2 shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with beta-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. RESULTS: The pretreatment with 25 dyne/cm2 but not with 0 or 1 dyne/cm2 shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that beta-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained alpha-smooth muscle actin were present. The thickness of this neointima averaged 41 +/- 12 micrometer and 60 +/- 23 micrometer in endothelial cell-seeded grafts that were pretreated with 25 dyne/cm2 shear stress and 1 dyne/cm2 shear stress, respectively, and 158 +/- 46 micrometer in grafts that were not seeded with endothelial cells. CONCLUSION: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness.

Expression of SET, an inhibitor of protein phosphatase 2A, in renal development and Wilms' tumor.

The human gene set was originally identified as a component of the set-can fusion gene produced by a somatic translocation event in a case of acute undifferentiated leukemia. In the developing kidney, set was highly expressed in the zone of nephron morphogenesis. Recently, SET was shown to be a potent and specific inhibitor of protein phosphatase 2A, a family of major serine/threonine phosphatases involved in regulating cell proliferation and differentiation. The current study sought to define further the role of SET in the regulation of renal cell proliferation and tumorigenesis. The mRNA encoding SET was expressed at much higher levels in transformed human and rodent cell lines than in cultured renal epithelial and primary endothelial cells. Consistent with a role for SET in cell proliferation, set mRNA expression was markedly reduced in cells rendered quiescent by serum starvation, contact inhibition, or differentiation. Previous findings during renal development were extended by demonstrating that SET protein expression is also much greater in developing rat and human kidney than in fully differentiated, mature kidney. Finally, high levels of set mRNA and SET protein expression were found in Wilms' tumor, but not in renal cell carcinoma, adult polycystic kidney disease or in transitional cell carcinoma.

Shear stress and the endothelium.

Shear stress and the endothelium. Vascular endothelial cells (ECs) in vivo are influenced by two distinct hemodynamic forces: cyclical strain due to vessel wall distention by transmural pressure, and shear stress, the frictional force generated by blood flow. Shear stress acts at the apical cell surface to deform cells in the direction of blood flow; wall distention tends to deform cells in all directions. The shear stress response differs, at least partly, from the cyclical strain response, suggesting that cytoskeletal strain alone cannot explain it. Acute shear stress in vitro elicits rapid cytoskeletal remodeling and activates signaling cascades in ECs, with the consequent acute release of nitric oxide and prostacyclin; activation of transcription factors nuclear factor (NF)kappaB, c-fos, c-jun and SP-1; and transcriptional activation of genes, including ICAM-1, MCP-1, tissue factor, platelet-derived growth factor-B (PDGF-B), transforming growth factor (TGF)-beta1, cyclooxygenase-II, and endothelial nitric oxide synthase (eNOS). This response thus shares similarities with EC responses to inflammatory cytokines. In contrast, ECs adapt to chronic shear stress by structural remodeling and flattening to minimize shear stress. Such cells become very adherent to their substratum and show evidence of differentiation. Increased adhesion following chronic shear stress has been exploited to generate vascular grafts with confluent EC monolayers, retained after implantation in vivo, thus overcoming a major obstacle to endothelialization of vascular prostheses.

Successful delayed bilateral renal revascularization during active phase of Takayasu's arteritis.

Successful bilateral renal revascularization was performed 24 days after the development of angiotensin converting enzyme-inhibitor-induced bilateral renal artery thrombosis and anuric acute renal failure in a patient with Takayasu's arteritis. Excellent results were obtained after an unusually long ischemic time for a patient with active-phase disease. The outcome suggests that aggressive surgical revascularization can benefit patients with renal failure caused by renal arterial occlusion during the active phase of Takayasu's arteritis.

TGF-beta type II receptor in rat renal vascular development: localization to juxtaglomerular cells.

To further define the role of transforming growth factor-beta (TGF-beta) receptors in renal vascular development, detailed immunohistochemical studies of TGF-beta receptor expression were performed from gestational day 15 through adulthood. On gestational day 15, TGF-beta type II receptor immunoreactivity was restricted to perirenal stromal and vascular cells. On gestational day 17 TGF-beta type II receptor immunoreactive stromal cells were observed within the kidney, with the same distribution as stromal alpha-smooth muscle actin and renin immunoreactive cells, and intense stromal TGF-beta type II receptor immunoreactivity continued through postnatal day 5. As vascular development progressed, TGF-beta type II receptor, alpha-smooth muscle actin and renin immunoreactivity became progressively restricted to small renal arteries and arterioles. Expression of TGF-beta type II receptors and renin was very intense in afferent glomerular arterioles during postnatal days 5 to 15, and then became progressively restricted only to juxtaglomerular cells in the mature kidney. TGF-beta type I receptor (ALK-5, ALK-1 and ALK-2) immunoreactivity was not detected in stromal or vascular elements during development or in the mature kidney. Intense TGF-beta type II receptor expression in renal stromal vascular smooth muscle cell precursors and developing blood vessels suggests a role for the TGF-beta type II receptors in the formation of the renal vascular smooth muscle compartment. The continued intense expression in juxtaglomerular cells argues for a role in renin synthesis and/or release. The absence of ALK-5, ALK-1, and ALK-2 in developing vascular smooth muscle and mature juxtaglomerular cells indicates that the canonical view of TGF-beta signaling may not hold in these locations.

Differential expression of transforming growth factor-beta receptors in rat kidney development.

Transforming growth factor-beta 1 (TGF-beta 1) is strongly expressed during embryogenesis and in sites undergoing intense development and morphogenesis. Two receptor serine/threonine kinases (types I and II) have been identified as signal-transducing TGF-beta receptors. This study was undertaken to further explore the role of the distinct TGF-beta receptors during kidney development. The species-specific sequence information for the two T beta R-I, namely, activin receptor-like kinase-5 (ALK-5) and Tsk7L, in the rat was sought. Two full-length T beta R-I cDNAs were cloned from a neonatal rat kidney and lung libraries, and sequencing revealed that they were the rat homologs of human ALK-5 and murine Tsk7L. Both types I and II TGF-beta receptors are expressed in the kidney as determined by Northern blot analysis. T beta R-II mRNA abundance was significantly greater in the neonatal rat kidney compared with the adult rat kidney. Similarly, ALK-5 mRNA was more highly expressed in the fetal and neonatal rat kidney than the adult rat kidney. In contrast, there was no significant difference in Tsk7L mRNA abundance among the fetal, neonatal, and adult rat kidney. Thus, based on these findings, both T beta R-II and ALK-5 are developmentally regulated in the kidney. Increased expression of T beta R-II and ALK-5 proteins in the developing kidney was confirmed by immunohistochemistry. Interestingly, the two TGF-beta receptors did not entirely colocalize, raising the intriguing possibility that other TGF-beta signaling receptors may be involved.

Smooth muscle cell proliferation in response to co-culture with venous and arterial endothelial cells.

PURPOSE: The critical role of endothelial cells (ECs) in arterial disease is well established, but little is known of their role in venous disease. Previous studies suggest inherent differences between arteries and veins: arterial stenoses demonstrate a large lipid component, whereas hemodialysis-related venous stenoses are characterized by marked smooth muscle cell (SMC) proliferation. This study compares effects of venous versus arterial ECs on SMC proliferation in co-culture. MATERIALS AND METHODS: Human saphenous vein ECs (HSV-ECs) or human aortic ECs (HA-ECs) were cultured on the underside of 10-micron, porous polycarbonate membranes and allowed to grow to confluence for 48 hours. After EC confluence, human aortic SMCs (HA-SMCs) were cultured on the membranes opposite the EC (day 0). On days 0, 1, 2, 4, 6, and 8, membranes were harvested (n = 3 per day), stained with Hoechst dye, and HA-SMCs were counted by fluorescence microscopy. Controls were HA-SMCs cultured alone. Comparisons were made by two-way multivariate analysis of variance. RESULTS: During the entire 8-day period, there was significant induction of HA-SMC proliferation by both HSV-ECs (P = .0003) and HA-ECs (P = .0012). Maximal inductions were 88% +/- 11% for HSV-ECs (P = .0015) and 24% +/- 6% for HA-ECs (P = .0015). HSV-ECs exhibited a three- to ninefold greater induction than HA-ECs (P = .0003). CONCLUSION: HSV-ECs induce adjacent HA-SMC proliferation, possibly in a paracrine manner to a significantly greater extent than HA-ECs.

TGF-beta and the endothelium during immune injury.

During immune injury, activation of endothelial cells by inflammatory cytokines stimulates leukocyte adhesion to the endothelium, turns the endothelium from an anticoagulant surface to one that is frankly procoagulant, and results in the release of vasoactive mediators and growth factors. Cytokine activation of endothelial cells also results in increased endothelial cell TGF-beta 1 synthesis and enhanced activation of latent TGF-beta, the latter involving a shift of plasmin production from the apical to subendothelial surface. In cytokine-stimulated endothelial cells, TGF-beta hinders leukocyte adhesion and transmigration via inhibition of IL-8 and E-selectin expression. TGF-beta also profoundly diminishes cytokine-stimulated inducible nitric oxide synthase production and instead augments endothelial nitric oxide synthase expression. Thus, some of the TGF-beta actions on endothelium during immune activation can viewed as immunosuppressive. TGF-beta also influences mechanisms of vascular remodeling during the healing phase of immune injury. It stimulates PDGF-B synthesis by endothelial cells, causes bFGF release from subendothelial matrix, and promotes VEGF synthesis by non-endothelial cells. Together these mediators control angiogenesis, a critical component of the vascular repair phenomenon. Further, endothelial cell derived PDGF-B and bFGF influence the proliferation and migration of neighboring cells. Thus, endothelial cells and TGF-beta actions on the endothelium play important roles both during the initial phase of immune injury and during the later remodeling phase.

Regulation of endothelial nitric-oxide synthase during hypoxia.

The mechanism by which nitric-oxide (NO) production increases during hypoxia is unknown. To explore the effect of hypoxia upon endothelial nitric-oxide synthase (ecNOS) activity and expression, we exposed bovine aortic endothelial cells (BAEC) to hypoxia (1% O2) for 0-24 h and measured levels of ecNOS mRNA, protein, and activity. The amount of ecNOS mRNA increases to more than twice the basal level after 6 h of hypoxia. Incubation of BAEC with actinomycin D during hypoxia prevents this increase, demonstrating that higher levels of mRNA observed during hypoxia are due to increased synthesis, not to increased stability of ecNOS mRNA. Levels of ecNOS protein increase throughout 24 h of hypoxia to more than twice normoxic levels. Although ecNOS expression increases within 2 h of hypoxia, total activity remains unchanged. To explore the transcriptional regulation of ecNOS, we constructed a reporter plasmid containing the ecNOS promoter region upstream of the luc gene and transfected this reporter plasmid into BAEC. In this system, hypoxia induces a linear increase over time in the expression of luciferase driven by the ecNOS promoter. It is concluded that hypoxia induces an increase in transcription of ecNOS in endothelial cells, activating the regulatory region of ecNOS by undefined transcription factors.

Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-beta receptors.

Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta-mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (T beta R-II) transdominant negative mutant. A mutant TGF-beta type II receptor (T beta R-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat T beta R-II cDNA as template. Since T beta R-II and TGF-beta type I receptor (T beta R-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type T beta R-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with T beta R-IIM, and four independent clones were expanded. That the T beta R-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface T beta R-IIM protein was shown by affinity cross-linking with 125I-TGF-beta 1. In wild-type endothelial cells, TGF-beta 1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 +/- 10% of control (n = 4). In transfected endothelial cells carrying T beta R-IIM, TGF-beta 1 stimulated [3H]thymidine incorporation to 131 +/- 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta 1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying T beta R-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with T beta R-II signaling inhibits this process in glomerular capillary endothelial cells.

Shear stress-conditioned, endothelial cell-seeded vascular grafts: improved cell adherence in response to in vitro shear stress.

BACKGROUND: Prosthetic vascular grafts with adherent endothelial cell monolayers may prove useful for small-caliber vessel bypass. However, endothelial cells adhere poorly to prosthetic graft material, and they are stripped when exposed to in vivo shear stress. This study sought to determine whether in vitro shear stress conditioning improves endothelial cell adhesion and decreases thrombogenicity of endothelial cell-seeded grafts. METHODS: The lumens of 1.5 mm (inside diameter) spun polyurethane polymer vascular grafts were seeded with bovine aortic endothelial cells and cultured in vitro for 6 days with or without continuous laminar shear stress, first at 1 to 2 dynes/cm2 for 3 days, then at approximately 25 dynes/cm2 for 3 days. Grafts preconditioned by shear stress and the static control grafts were then exposed to arterial shear stress at 25 dynes/cm2 for 25 seconds. The number of dislodged cells was counted, and the grafts were examined by light and scanning electron microscopy. Whole blood clotting time in the grafts was also determined. RESULTS: Exposure of grafts to acute shear stress dislodged 1.35 x 10(6) +/- 0.44 x 10(6) cells from static grafts compared with 1.05 x 10(4) +/- 0.16 x 10(4) cells from grafts preconditioned by shear stress. By light and electron microscopy an intact endothelial monolayer was observed to cover the lumen of shear stress-conditioned grafts, whereas few cells remained on the luminal surface of grafts not previously exposed to shear stress. The clotting time in shear stress-conditioned grafts was significantly prolonged in relation to grafts not exposed to shear stress. CONCLUSIONS: These findings show that endothelial cell adhesion and retention on vascular grafts in vitro is markedly enhanced by preconditioning the seeded endothelial cell monolayer with long-term shear stress. Consequently, vascular grafts containing shear stress-conditioned endothelial monolayers are less thrombogenic in vitro than small-caliber vascular grafts without intact endothelial cell monolayers.