Browsing by Subject "Endothelium, Vascular"
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Item Histone Deacetylase 7 and Transcriptional Regulatory Networks of the Vascular Endothelium(2010-05-14) Young, Bryan Daniel; Olson, EricCells respond to stimuli in part through the modulation of gene expression. Signal transduction from the environment to the nucleus culminates in the activation of factors that modify chromatin structure to either facilitate or inhibit gene transcription. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two such classes of enzymes that regulate the epigenetic code. Their opposing actions – to activate transcription by histone acetylation and to inhibit transcription by deacetylation – are tightly regulated to coordinate the vast gene programs required for cellular growth and differentiation. The class II HDACs are restricted in their expression patterns, and each have unique developmental and physiological functions. The studies described here focus on HDAC7, a class II HDAC that is expressed in vascular endothelial cells and whose function is essential for the maintenance of vascular integrity during embryogenesis. Mice lacking HDAC7 die by e11.5 with complex cardiovascular malformations including endothelial, vascular smooth muscle, and myocardial defects. By generating HDAC7 conditional knockout mice, it was observed that all of these defects are recapitulated in mice bearing an endothelial-specific deletion of HDAC7, but no defects are observed upon deletion of HDAC7 in the other cell types that were affected in the HDAC7 nulls. This in vivo evidence demonstrated that HDAC7 acts cell autonomously to maintain normal vascular development, and lead to the identification of the genetic abnormalities and mechanism leading to cardiovascular failure in the HDAC7 knockout. Further, this work begins the investigation of HDAC7 in adult vascular physiology, the findings of which will reveal new mechanisms whereby the vasculature responds to stress signals or disease. To this end, methods have been developed for the deletion of HDAC7 in the adult mouse using an inducible cre recombinase system together with the HDAC7 conditional allele. Additionally, these studies present progress toward the identification of the enhancer elements driving the endothelial-specific expression pattern of HDAC7. Detailed characterization of this enhancer is likely to implicate new signaling pathways as being involved in the genetic regulation of vascular development and maintenance. Finally, this work investigates the role of microRNAmediated gene silencing in the vascular system by identifying microRNAs involved in MEF2-dependent signaling in endothelial cells.Item Mechanisms of Intradialytic Hypertension(2012-08-15) Kim, Bohyun Catherine; Inrig, JulaIntradialytic Hypertension and Its Association with Endothelial Cell Dysfunction Background: Intradialytic hypertension is associated with adverse outcomes, yet the mechanism is uncertain. Patients with intradialytic hypertension exhibit imbalances in endothelial-derived vasoregulators nitric oxide and endothelin-1, indirectly suggesting endothelial cell dysfunction. We hypothesized that intradialytic hypertension is associated in vivo with endothelial cell dysfunction, a novel predictor of adverse cardiovascular outcomes. Design, settings, participants, & measurements: We performed a case-control cohort study including 25 hemodialysis (HD) subjects without (controls) and 25 with intradialytic hypertension (an increase in systolic BP pre- to postdialysis greater than or equal to 10 mmHg greater than or equal to 4/6 consecutive HD sessions). The primary outcome was peripheral blood endothelial progenitor cells (EPCs) assessed by aldehyde dehydrogenase activity (ALDHbr) and cell surface marker expression (CD34+CD133+). We also assessed endothelial function by ultrasonographic measurement of brachial artery flow-mediated vasodilation (FMD) normalized for shear stress. Parametric and nonparametric t tests were used to compare EPCs, FMD, and BP. Results: Baseline characteristics and comorbidities were similar between groups. Compared with controls, 2-week average predialysis systolic BP was lower among subjects with intradialytic hypertension (144.0 versus 155.5 mmHg), but postdialysis systolic BP was significantly higher (159.0 versus 128.1 mmHg). Endothelial cell function was impaired among subjects with intradialytic hypertension as measured by decreased median ALDHbr cells and decreased CD34+CD133+ cells (ALDHbr, 0.034% versus 0.053%; CD34+CD133+, 0.033% versus 0.059%). FMD was lower among subjects with intradialytic hypertension (1.03% versus 1.67%). Conclusions: Intradialytic hypertension is associated with endothelial cell dysfunction. We propose that endothelial cell dysfunction may partially explain the higher event rates observed in these patients. Probing the Mechanisms of Intradialytic Hypertension: a Pilot Study Targeting Endothelial Cell Dysfunction Background: Intradialytic hypertension may be caused by an impaired endothelial cell response to hemodialysis. Carvedilol has been shown to improve endothelial cell function in vivo and in vitro to block endothelin-1 release. Among patients with intradialytic hypertension, we hypothesized that carvedilol would improve endothelial cell function and reduce the occurrence of intradialytic hypertension. Design, settings, participants & measurements: We performed a prospective 12-week pilot study of carvedilol titrated to 50 mg twice daily among 25 hemodialysis participants with intradialytic hypertension. Each patient served as their own control. Changes in endothelial cell function (assessed by flow-mediated vasodilation, endothelial progenitor cells (EPCs by aldehyde dehydrogenase activity and CD34+CD133+), asymmetric dimethylarginine (ADMA) and endothelin-1) and blood pressure (BP) from baseline to study-end were analyzed by paired tests. Results: Flow-mediated vasodilation was significantly improved with carvedilol (from 1.03% to 1.40%, p=0.02). There was no significant change in EPCs, endothelin-1 or ADMA. At baseline, participants exhibited a significant increase in endothelin-1 pre to postdialysis that resolved by study-end. While pre-hemodialysis systolic BP was unchanged (144 to 146 mmHg, p=0.5), post-hemodialysis systolic BP, 44-hour ambulatory systolic BP, and the frequency of intradialytic hypertension decreased with carvedilol (159 to 142 mmHg, p<0.0001; 155 to 148 mmHg, p=0.05; 77% (4.6/6) to 28% (1.7/6), p<0.0001, respectively). Conclusions: Among hemodialysis participants with intradialytic hypertension, targeting endothelial cell dysfunction with carvedilol was associated with modest improvements in endothelial cell function, improved intra and interdialytic BP, and reduced frequency of intradialytic hypertension. Randomized controlled trials are required to confirm these findings. The Role of Dialysate Exposure in Intradialytic Hypertension Background: Intradialytic hypertension is associated with endothelial dysfunction, but the cause of vascular impairment is unknown. Exposure to high concentration sodium has been shown in vitro to promote endothelial stiffness and imbalances in markers of vascular function. We hypothesized that, among patients with intradialytic hypertension, exposure to dialysate sodium would lead to increases in endothelin-1, decreases in nitric oxide, and an intradialytic increase in systolic blood pressure. Design, settings, participants & measurements: We performed a 6-week crossover study of 10 hemodialysis patients with intradialytic hypertension. Changes in blood pressure, endothelin-1, and nitric oxide levels were measured during three different, midweek dialysis treatments consisting of: 1) regular hemodialysis with standard dialysate sodium (140 mEq/L); 2) ultrafiltration only without dialysate exposure; and 3) hemodialysis (Na 140 mEq/L) without ultrafiltration. These changes were analyzed using mixed model analyses. Results: Serum sodium levels rose with dialysate exposure during regular HD and HD without UF sessions (+1.6 and +3 mEq/L, respectively), and fell during UF only session (-0.9 mEq/L). Endothelin-1 level also rose with dialysate exposure during regular HD and HD without UF (+0.15 and +0.25pg/mL, respectively), but fell during UF only session (-0.02 pg/mL). Plasma nitrite levels fell with all treatment types, most significantly with regular HD (-123.25 nM), then HD without UF (-52.77 nM), with lowest decrease seen during UF only session without dialysate exposure (-48.48 nM). Systolic BP rose during all treatments, most significantly with HD without UF (13.3%), followed by regular dialysis (6.9%), and UF only (5.7%). Conclusions: Among hemodialysis patients prone to intradialytic hypertension, there was an association between dialysate exposure and increases in endothelin-1, decreases in nitric oxide, and increases in systolic blood pressure during dialysis. We propose that high dialysate to plasma sodium gradient may contribute to intradialytic hypertension. [Keywords: hemodialysis; intradialytic hypertension; Carvedilol; endothelial cell dysfunction; endothelial progenitor cells]Item Targeting Aminophospholipids Exposed On Tumor Endothelium for Tumor Imaging(2012-07-20) Stafford, Jason Hugh; Thorpe, Philip E.Advances in noninvasive imaging of human cancer are crucial to improving diagnosis and therapeutic planning. My project was aimed at developing novel imaging agents that target the aminophospholipidsphosphatidylserine (PS) and phosphatidylethanolamine (PE). PS and PE arenormally intracellular, but become exposed on the surface of tumor endothelial cells (EC). Anti-tumor therapies promote exposure of PS and PE on tumor EC and the tumor cells as well. Therefore, I tested the hypothesis that 1N11, a PS-binding antibody, and duramycin, a PE-binding peptide, could function as tumor imaging agents. I labeled the F(ab’)2 fragment of 1N11 with the near-infrared fluophore 800CW for optical imaging and the positron emitting isotope iodine-124 (124I) for PET imaging. 800CW-1N11 F(ab’)2 clearly imaged subcutaneous and orthotopic U87 gliomas growing in mice with optimal tumor contrast obtained at 24 h post-injection (p.i.). Uptake of 800CW-1N11 F(ab’)2 was approximately 2-fold higher in irradiated U87 tumors. 124I-1N11 F(ab’)2 clearly imaged subcutaneous and orthotopic PC3 prostate carcinomas growing in mice with optimal tumor contrast obtained at 48 hr p.i. Importantly, 800CW- and 124I-1N11 F(ab’)2 exhibited low uptake in non-target organs (i.e. liver and kidneys). Unlike PS, PE had not been established as a specific marker of tumor vasculature in the literature. To demonstrate PE was such a marker, I biotinylated duramycin, characterized its binding properties, and used it to determine the distribution of PE on EC in vitro and in vivo. Exposure of cultured EC to hypoxia, acidity, reactive oxygen species, or irradiation resulted in the formation of membrane blebs that were intensely PE-positive. When biotinylated duramycin was intravenously injected into tumor-bearing mice it preferentially localized to the luminal surface of the vascular endothelium in multiple tumor models. PE-positive vessels were observed in and around hypoxic regions of the tumor. With the exception of intertubular vessels of the kidney, normal vessels remained unstained. I also conjugated duramycin to 800CW and used it for optical imaging of RM-9 and TRAMP prostate carcinomas. These results demonstrate that both 1N11 and duramycin can be used to image a variety of tumors and warrant further study as imaging agents. [Keywords" cancer, imaging, phospholipids, antibodies, peptides, optical, PET, phosphatidylserine, phosphatidylethanolamine, vasculature]