Identification of a potent endothelium-derived angiogenic factor.

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up4U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up4U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up4U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up4U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up4U after stimulation with shear stress. Mean total plasma Up4U concentrations of healthy subjects (N=6) were sufficient to induce angiogenic and proliferative effects (1.34 ± 0.26 nmol L(-1)). In conclusion, Up4U is a novel strong human endothelium-derived angiogenic factor.

Uridine adenosine tetraphosphate acts as an autocrine hormone affecting glomerular filtration rate.

Recently, uridine adenosine tetraphosphate (Up(4)A) was described as a strong vasoconstrictor released from endothelial cells after stimulation with mechanical stress. In this study, we isolated and identified Up(4)A from kidney tissue, and we characterized the essential varying effects of Up(4)A on the afferent and efferent arterioles. Porcine and human kidney tissue was fractionated by size exclusion chromatography, affinity chromatography, anion exchange chromatography and reverse phase chromatography. In fractions purified to homogeneity, Up(4)A was identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), MALDI-LIFT fragment mass spectrometry (MALDI-TOF/TOF MS), retention-time comparison and enzymatic cleavage analysis. We analysed the release of Up(4)A from cultivated renal proximal tubule cells after stimulation of protein kinase C with oleoyl-2-acetyl-sn-glycerol (OAG). Up(4)A was identified in renal tissue, and the effect of Up(4)A on the vascular tone of isolated perfused afferent and efferent arterioles was tested. Stimulation of tubule cells with OAG increased the release rate of Up(4)A from tubule cells about tenfold. Up(4)A acts as a strong vasoconstrictive mediator on afferent arterioles, but has no significant effect on the tone of efferent arterioles, suggesting a functional role of Up(4)A as an autocrine hormone for glomerular perfusion. Because of the predominant effect of the Up(4)A on afferent arterioles, we assume that Up(4)A may decrease glomerular perfusion, intra-glomerular pressure and, hence, glomerular filtration rate. The release of Up(4)A from renal tubular cells may be an additional mechanism whereby tubular cells could affect renal perfusion. Up(4)A release may further contribute to renal vascular autoregulation mechanisms. In conclusion, as Up(4)A occurs in renal tissue and has marked effects on afferent but not efferent arterioles, Up(4)A may play a role in renal hemodynamics and possibly blood pressure regulation.

Increased uridine adenosine tetraphosphate concentrations in plasma of juvenile hypertensives.

BACKGROUND: Uridine adenosine tetraphosphate (Up4A) was been recently characterized as a potent vasoconstrictor. Up4A occurs in plasma from healthy subjects at concentrations sufficient to cause strong vasoconstrictive effects. In this study, Up4A concentrations in plasma from juvenile hypertensives and normotensives were determined. METHODS AND RESULTS: Up4A was purified to homogeneity by preparative reverse phase high performance liquid-chromatography (HPLC), affinity chromatography HPLC, and analytic reverse phase HPLC from deproteinized plasma of juvenile hypertensives and normotensives. Mean total plasma Up4A concentration was significantly increased in juvenile hypertensives compared with juvenile normotensives (33.0+/-25.4 versus 3.7+/-0.9 nmol/L; mean+/-SEM, n=40 and 38, respectively; P<0.005). Accordingly, Up4A showed a significant association with juvenile hypertension (OR for ln(Up4A): 1.82; 95% CI 1.12, 2.95). Plasma Up4A concentrations correlated with left ventricular mass (Kendall-tau correlation coefficient 0.220, n=40; P<0.05) and intima media wall thickness (Kendall-tau correlation coefficient 0.296, n=40; P<0.05) in the hypertensives. Because the increased intima media thickness may be related to proliferative effects of Up4A, we studied the effects of Up4A on human vascular smooth muscle cell proliferation. The maximum proliferative effect of Up4A was 80.0+/-24.0% % above control (P<0.01). The proliferative effect of Up4A on smooth muscle cells is cell cycle-dependent, involving stimulation of S phase entry. CONCLUSION: Circulating levels of Up4A are strongly associated with juvenile hypertension. The endothelium-derived vasoconstrictor Up4A may contribute to the early development of primary hypertension and is moreover an important risk factor of juvenile hypertension.

Mass-spectrometric identification of a novel angiotensin peptide in human plasma.

OBJECTIVE: Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. METHODS AND RESULTS: Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A/Ang II was higher in end-stage renal failure patients. CONCLUSIONS: Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.