The vascular-disrupting agent, combretastatin-A4-phosphate, enhances neurogenic vasoconstriction in rat small arteries.

Combretastatin-A4-phosphate (CA4P/CA4), an anti-cancer drug, induces tumour hypoxia by destabilizing the cytoskeleton in tumour endothelial cells. Hypertensive side effects have been observed. We hypothesized that CA4P/CA4 lead to endothelial dysfunction followed by increased vasoconstriction. Mesenteric small arteries and femoral arteries isolated from male Wistar rats were mounted in microvascular myographs for isometric tension recordings and electrical field stimulation (EFS). Immunoblotting of endothelial nitric oxide synthase (eNOS) was performed on human umbilical vein endothelial cells (HUVECs). CA4P failed per se to change vascular tone. In femoral arteries, endothelial cell removal, l-nitro-arginine (l-NNA, an inhibitor of eNOS) and CA4P enhanced phenylephrine-induced vasoconstriction, while in mesenteric arteries only l-NNA leftward shifted concentration-response curves for phenylephrine. CA4P enhanced vasoconstriction induced by low frequency (0.5-4Hz) EFS in femoral arteries, but not in mesenteric arteries. Neurogenic contractions were inhibited by prazosin, an α(1)-adrenoceptor antagonist. In mesenteric arteries, CA4P and l-NNA inhibited vasorelaxation induced by vanadate, a tyrosine phosphatase inhibitor. CA4P did not affect acetylcholine-induced relaxation. In HUVECs, CA4P increased phosphorylation at eNOS-Thr(495), a negative regulatory site, while the positive phosphorylation site eNOS-Ser(1177) was not affected. CA4 neither influenced the actions of phenylephrine, vanadate nor acetylcholine in femoral and mesenteric arteries. In conclusion, our findings suggest that CA4P, but not CA4, enhances sympathetic adrenergic vasoconstriction probably by increasing eNOS-Thr(495) phosphorylation, in a tissue selective manner. These findings encourage further investigation to show that the hypertension and regional organ ischemia induced by CA4P can be avoided by concomitant treatment with an α(1)-adrenoceptor antagonist.

Pulmonary pressure reduction attenuates expression of proteins identified by lung proteomic profiling in pulmonary hypertensive rats.

The present study was designed to analyze protein expression in lungs from pulmonary hypertensive rats in order to identify novel signaling pathways. This was achieved by proteomic studies in which proteins from lung homogenates from hypoxic were compared to normoxic rats. The expression of these proteins was then investigated in lungs from hypoxic rats treated with either an activator of soluble guanylyl cyclase, BAY 412272, or an inhibitor of phosphodiesterase type 5, sildenafil. The proteomic study revealed an up-regulation of guanine nucleotide-binding protein ß, GST-ω-1, cathepsin D, chloride intracellular channel subunit 5, annexin A4, F-actin capping protein CapZ (CapZα), and the translation factor elongation factor 1 δ in lungs from chronic hypoxic rats with pulmonary hypertension. Immunohistochemistry revealed that CapZα, cathepsin D, and annexin A4 were expressed in the pulmonary vascular wall and immunoblotting showed these proteins correlated to alterations in muscularization. Both drugs inhibited hypoxia-induced increase in right ventricular systolic pressure and pulmonary arterial muscularization, and prevented most of the protein regulations observed after hypoxia. These findings suggest that pulmonary pressure is an important factor for initiating signaling pathways leading to protein expression and muscularization in the pulmonary vasculature.

BAY 41-2272 inhibits the development of chronic hypoxic pulmonary hypertension in rats.

The present study investigated whether BAY 41-2272(5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine), a novel pyrazolopyridine that activates guanylyl cyclase and sensitizes the enzyme towards nitric oxide (NO), inhibits the development of pulmonary hypertension. BAY 41-2272 (1 or 10 mg/kg/day) was administered intraperitoneally, and sildenafil (25 mg/kg/day), an inhibitor phosphodiesterase type 5, was given in the drinking water to rats kept under chronic hypobaric hypoxia for two weeks. Right ventricular systolic pressure and hypertrophy, degree of muscularization and relaxation of pulmonary arteries were measured, and immunoblotting was performed. Chronic hypoxia increased right ventricular systolic pressure and expression of soluble guanylyl cyclase and phosphorylated vasodilator-stimulated phosphoprotein (VASP-P(ser239)). BAY 41-2272 prevented hypoxia-induced increase in right ventricular systolic pressure and right ventricular hypertrophy to the same extent as sildenafil. Only sildenafil significantly decreased hypoxia-induced muscularization of pulmonary arteries. Expressed relative to soluble guanylyl cyclase expression, VASP-P(ser239) was increased in lungs from rats treated with BAY 41-2272. Acutely BAY 41-2272 caused pulmonary as well as systemic vasodilatation. In the chronic setting systemic blood pressure was not different to baseline at trough after intraperitoneally administered BAY 41-2272. BAY 41-2272 vasorelaxation in isolated pulmonary resistance arteries was inhibited by an inhibitor of guanylyl cyclase, ODQ (1H-[1,2,4] oxadiazolo[4,3-a]quinoxaline-1-one), and of Na(+)-K(+)-ATPase, ouabain. In conclusion, in an adult rat model of chronic hypoxic pulmonary hypertension, BAY 41-2272 to a similar degree as sildenafil prevents pulmonary hypertension. Thus, BAY 41-2272 may provide a novel therapeutic compound for treating chronic hypoxic pulmonary hypertension.

Proteomics reveals lowering oxygen alters cytoskeletal and endoplasmatic stress proteins in human endothelial cells.

A proteomic approach was applied to explore the signalling pathways elicited by lowering O(2) in endothelial cells. Endothelial cells isolated from native umbilical cords were subjected to 21, 5, or 1% O(2) for 24 h. 2-D PAGE was performed and candidate proteins were identified using LC-MS/MS. Lowering of O(2) from 21 to 5% induced upregulation of cofilin-1, cyclophilin A, tubulin and tubulin fragments, a fragment of glucose-regulated protein 78 (Grp78) and calmodulin. The upregulation of Grp78 suggested that ER stress proteins were altered and indeed Grp94 and caspase 12 expression were increased in cells exposed to 5% O(2). The presence of ER stress is also supported by findings of blunted caffeine-evoked ER calcium release in cells exposed to 5 and 1% O(2). Exposure to 1% O(2) caused increases in cofilin-1, cyclophilin A, and caspase 12 as well as a decrease of beta-actin, but it did not alter the expression of calmodulin, tubulin, Grp78, and Grp94. Incubation with CoCl(2), a stabilizer of the hypoxia-inducible factor, increased the expression of several of the proteins. The present investigations reveal that lowering O(2), probably in part through hypoxia-inducible factor, alter the expression of a series of proteins mainly involved in cytoskeletal changes (e.g. cofilin-1, tubulin, and beta-actin) and in ER stress/apoptosis (e.g. Grp78/94, caspase 12, and cyclophilin A).

Diminished NO release in chronic hypoxic human endothelial cells.

The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 microM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser(1177) and eNOS Thr(495) phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser(1177), was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of beta- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.

MAP kinases and heat shock-induced hormesis in human fibroblasts during serial passaging in vitro.

Adult human skin fibroblasts were exposed repeatedly to 41 degrees C or 42 degrees C heat shock (HS) for 1 h twice a week during serial passaging throughout their replicative life span. On the basis of longevity curves, cell size, and morphology, we observed that repeated mild heat shock (RMHS) at 41, degrees C had strong anti-aging hormetic effects, including 20% extension of cellular longevity. The basal levels of the MAP kinases JNK1, JNK2, and p38 increased during serial passaging, while that of ERK2 decreased. RMHS further exaggerated these effects, which suggests that age-related changes in MAP kinases may be an adaptive response for better cell survival.

Molecular mechanisms of anti-aging hormetic effects of mild heat stress on human cells.

In a series of experimental studies we have shown that repetitive mild heat stress has anti-aging hormetic effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. We have reported the hormetic effects of repeated challenge at the levels of maintenance of stress protein profile; reduction in the accumulation of oxidatively and glycoxidatively damaged proteins; stimulation of the proteasomal activities for the degradation of abnormal proteins; improved cellular resistance to ethanol, hydrogenperoxide, and ultraviolet-B rays; and enhanced levels of various antioxidant enzymes. We are now undertaking a detailed analysis of the signal transduction pathways to determine alterations in the phosphorylation and dephosphorylation states of extracellular signal-related kinase, c-Jun terminal kinase and p38 MAP-kinases as a measure of cellular responsiveness to mild and severe heat stress. Furthermore, we are also undertaking comparative studies using non-aging immortal cell lines, such as SV40-transformed human fibroblasts, spontaneous osteosarcoma cells, and telomerase-immortalized human bone marrow cells for establishing differences in normal and cancerous cells with respect to their responsiveness to mild and severe stresses.

Pressure-induced activation of extracellular signal-regulated kinase 1/2 in small arteries.

Extracellular signal-regulated kinase 1/2 (ERK1/2) may play a central signaling role in vascular remodeling. We investigated a possible combined role for the renin-angiotensin system and platelet-derived growth factor beta-receptor (PDGF-beta-R) in pressure-induced ERK1/2 activation in intact rat mesenteric small arteries. In an organ culture model, vessels were pressurized (70 mm Hg) for 1 hour plus a 5-minute intervention period. The intervention was either a rise in intraluminal pressure (up to 140 mm Hg) or challenge with angiotensin II (Ang II, 0.1 micromol/L) or PDGF-BB (30 microg/L). ERK1/2 activation was determined by Western blotting as formation of phosphorylated ERK1/2. All interventions caused ERK1/2 activation that was inhibited by the MEK inhibitor PD98059. The response to pressure was inhibited by an ACE inhibitor (perindoprilat), an Ang II receptor type 1 (R-AT1) antagonist (candesartan), and tyrosine kinase inhibitors (genistein, herbimycin A). An R-AT2 antagonist (PD123319) had no significant effect. Both a PDGF-receptor tyrosine kinase inhibitor (RPR101511A) and a neutralizing PDGF-beta-R antibody (AF385) inhibited the activation of ERK1/2 caused by PDGF-BB, Ang II, and pressure. That the latter interventions could indeed inhibit the PDGF-beta-R was supported by experiments with unmounted vessels in which PDGF-beta-R activation was measured by Western blot; both PDGF-BB and Ang II-mediated PDGF-beta-R activation were inhibited by RPR101511A and AF385. Immunohistochemistry showed that ERK1/2 and PDGF-beta-R was located in the adventitia, tunica media, and intima. The results suggest that pressure in rat mesenteric small arteries causes acute activation of ERK1/2 through pathways involving Ang II and PDGF-beta-R.