To understand the role of TGF-β signaling in cardiovascular development, we generated mice with conditional deletion of the TGF-β type II receptor (TβRII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22α. The SM22α promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and myocardium. All SM22-Cre(+/-)/Tgfbr2 (flox/flox) embryos died during the last third of gestation. About half the mutant embryos exhibited heart defects (ventricular myocardium hypoplasia and septal defects). All mutant embryos displayed profound vascular abnormalities in the descending thoracic aorta (irregular outline and thickness, occasional aneurysms and elastic fiber disarray). Restriction of these defects to the descending thoracic aorta occurred despite similar levels of Tgfbr2 invalidation in the other portions of the aorta, the ductus arteriosus and the pulmonary trunk. Immunocytochemistry identified impairment of VSMC differentiation in the coronary vessels and the descending thoracic aorta as crucial for the defects. Ventricular myocardial hypoplasia, when present, was associated to impaired α-SMA differentiation of the epicardium-derived coronary VSMCs. Tgfbr2 deletion in the VSMCs of the descending thoracic aorta diminished the number of α-SMA-positive VSMC progenitors in the media at E11.5 and drastically decreased tropoelastin (from E11.5) and fibulin-5 (from E.12.5) synthesis and/or deposition. Defective elastogenesis observed in all mutant embryos and the resulting dilatation and probable rupture of the descending thoracic aorta might explain the late embryonic lethality. To conclude, during mouse development, TGF-β plays an irreplaceable role on the differentiation of the VSMCs in the coronary vessels and the descending thoracic aorta.

In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.

We have previously reported the presence and the persistence of immunoreactive GnRH cells in the rat anterior pituitary in the absence of exogenous GnRH. To substantiate the hypothesis of its endogenous production, we have investigated the presence of GnRH mRNA in rat anterior pituitary tissue using a reverse transcription-polymerase chain reaction (RT-PCR) amplification protocol. Total RNA from rat anterior pituitary, hypothalamus (positive control), and muscle (negative control) was reverse transcribed to the first strand of cDNA and amplified by PCR using a set of three exon-specific primers for a target sequence of the GnRH gene, i.e. two external 5' and 3' primers and one internal 3' primer. UV light analysis of the size-fractionated RT-PCR products showed two bands of the predicted sizes [511 and 397 base pairs (bp)] hybridizing with GnRH probes in Southern analysis only in hypothalamus and in anterior pituitary but not in muscle. These two bands were far more intense in the hypothalamus than in the anterior pituitary. Restriction enzyme analysis evidenced that the two RT-PCR products included the HindIII endonuclease site of cleavage present in the GnRH cDNA sequence spanned by the primers, yielding two digested products of the anticipated sizes (86 and 425 bp for the 511-bp fragment, and 86 and 311 bp for the 397-bp fragment). The complementary sequences for the GnRH probes were conserved in the 425-bp and 311-bp fragments. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target GnRH sequences in the anterior pituitary as well as in the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: TGF-β shifts from inhibition to stimulation of vascular smooth muscle cell (vSMC) growth when cell density increases. How proliferation and apoptosis contribute to this shift is still unknown. Methods: In sparse and confluent V8 vSMC treated or not with TGF-β₁ (1 ng/ml) for 3 days, cell number, mitotic activity, cell-cycle-regulatory protein levels, caspase-3 and phosphoinositide 3-kinase (PI3-K) activities were studied. Results: In TGF-β₁-treated cells, (i) the growth curve rose constantly compared to controls, reaching post-confluent densities; (ii) mitotic activity, which was constant at all cell densities, was lower than in sparse but higher than in contact-inhibited control cells, and (iii) apoptosis occurred at sparse densities only. The mechanism of proliferation control by TGF-β₁ was very unconventional in V8 vSMCs: (i) p15^INK4b and cyclin D levels were similar in cells treated or not with TGF-β₁, and (ii) p27^Kip1 levels remained very low even at high densities while cyclin E levels were not markedly decreased. TGF-β₁-induced apoptosis in sparse cultures and its reversal in dense cultures were inversely correlated to PI3-K activation. Conclusions: TGF-β₁ slowed sparse V8 vSMC growth by inhibiting proliferation and inducing apoptosis. TGF-β₁-treated confluent vSMCs escaped contact inhibition and kept growing through unconventional regulation of p27^Kip1, cyclin E and suppression of apoptosis.