Induction of cell cycle-dependent genes during cell cycle progression of arterial smooth muscle cells in culture.

Serum stimulation of arterial smooth muscle cells in culture induces a progression through the cell cycle and cell proliferation. Most genes previously described as cell cycle-dependent in various cell types also demonstrate a cell cycle-dependent expression in arterial smooth muscle cells. As in other cell types, these genes can be classified into three groups according to their mode of expression: "immediate early" genes (c-fos, c-myc, ...), "delayed early" genes (2F1, ...), and "late-G1" genes (proliferating cell nuclear antigen, thymidine kinase, . . .). In addition to these previously described genes, three genes isolated from a cDNA library of stimulated smooth muscle cells have been demonstrated to be cell cycle-dependent: A21, the rat JE gene, and L51 can be classified as "immediate early" genes, while M11 represents a new member of the "delayed early" gene family.     

Biopterin Metabolism and eNOS Expression during Hypoxic Pulmonary Hypertension in Mice

Tetrahydrobiopterin (BH₄), which fosters the formation of and stabilizes endothelial NO synthase (eNOS) as an active dimer, tightly regulates eNOS coupling / uncoupling. Moreover, studies conducted in genetically-modified models demonstrate that BH₄ pulmonary deficiency is a key determinant in the pathogenesis of pulmonary hypertension. The present study thus investigates biopterin metabolism and eNOS expression, as well as the effect of sepiapterin (a precursor of BH₄) and eNOS gene deletion, in a mice model of hypoxic pulmonary hypertension. In lungs, chronic hypoxia increased BH₄ levels and eNOS expression, without modifying dihydrobiopterin (BH₂, the oxidation product of BH₄) levels, GTP cyclohydrolase-1 or dihydrofolate reductase expression (two key enzymes regulating BH₄ availability). In intrapulmonary arteries, chronic hypoxia also increased expression of eNOS, but did not induce destabilisation of eNOS dimers into monomers. In hypoxic mice, sepiapterin prevented increase in right ventricular systolic pressure and right ventricular hypertrophy, whereas it modified neither remodelling nor alteration in vasomotor responses (hyper-responsiveness to phenylephrine, decrease in endothelium-dependent relaxation to acetylcholine) in intrapulmonary arteries. Finally, deletion of eNOS gene partially prevented hypoxia-induced increase in right ventricular systolic pressure, right ventricular hypertrophy and remodelling of intrapulmonary arteries. Collectively, these data demonstrate the absence of BH₄/BH₂ changes and eNOS dimer destabilisation, which may induce eNOS uncoupling during hypoxia-induced pulmonary hypertension. Thus, even though eNOS gene deletion and sepiapterin treatment exert protective effects on hypoxia-induced pulmonary vascular remodelling, increase on right ventricular pressure and / or right ventricular hypertrophy, these effects appear unrelated to biopterin-dependent eNOS uncoupling within pulmonary vasculature of hypoxic wild-type mice.     

A new series of M3 muscarinic antagonists based on the 4-amino-piperidine scaffold

A series of 4-amino-piperidine containing molecules have been synthesized and structure-affinity relationship toward the M3-muscarinic receptor has been investigated. Chemical modulations provided molecules with K(i) for the human M3-R up to 1 nM with variable selectivity (3- to 40-fold) over the human M2-R. Compounds 2 (pA(2)=8.3, 8.6) demonstrates in vitro on guinea pig bladder and ileal strips potent anticholinergic properties and tissue selectivity.     

Estradiol accelerates endothelial healing through the retrograde commitment of uninjured endothelium

Although the accelerative effect of 17beta-estradiol (E2) on endothelial regrowth has been clearly demonstrated, the local cellular events accounting for this beneficial vascular action are still uncertain. In the present work, we compared the kinetics of endothelial healing of mouse carotid arteries after endovascular and perivascular injury. Both basal reendothelialization as well as the accelerative effect of E2 were similar in the two models. Three days after endothelial denudation, a regenerative area was observed in both models, characterized by similar changes in gene expression after injury, visualized by en face confocal microscopy (EFCM). A precise definition of the injury limits was only possible with the perivascular model, since it causes a complete and lasting decellularization of the media. Using this model, we demonstrated that the migration of uninjured endothelial cells precedes proliferation (bromodeoxyuridine incorporation) and that these events occur at earlier time points with E2 treatment. We have also identified an uninjured retrograde zone as an intimate component of the endothelial regeneration process. Thus, in the perivascular model, the regenerative area can be subdivided into a retrograde zone and a reendothelialized area. Importantly, both areas are significantly enlarged by E2. In conclusion, the combination of the electric perivascular injury model and EFCM is well adapted to the visualization of the endothelial monolayer and to investigate cellular events involved in reendothelialization. This process is accelerated by E2 as a consequence of the retrograde commitment of an uninjured endothelial zone to migrate and proliferate, contributing to an enlargement of the regenerative area.     

Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.     

Time course of osteopontin, osteocalcin, and osteonectin accumulation and calcification after acute vessel wall injury.

Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.     

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.     

Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.