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.

Normal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy. Recently, plasminogen activator inhibitor type 1 (SERPINE1/PAI-1) was reported as an essential mediator of late radiation-induced intestinal injury. However, it is not clear whether PAI-1 plays a role in acute radiation-induced intestinal damage and we hypothesized that PAI-1 may play a role in the endothelium radiosensitivity. In vivo, in a model of radiation enteropathy in PAI-1 −/− mice, apoptosis of radiosensitive compartments, epithelial and microvascular endothelium was quantified. In vitro, the role of PAI-1 in the radiation-induced endothelial cells (ECs) death was investigated. The level of apoptotic ECs is lower in PAI-1 −/− compared with Wt mice after irradiation. This is associated with a conserved microvascular density and consequently with a better mucosal integrity in PAI-1 −/− mice. In vitro, irradiation rapidly stimulates PAI-1 expression in ECs and radiation sensitivity is increased in ECs that stably overexpress PAI-1, whereas PAI-1 knockdown increases EC survival after irradiation. Moreover, ECs prepared from PAI-1 −/− mice are more resistant to radiation-induced cell death than Wt ECs and this is associated with activation of the Akt pathway. This study demonstrates that PAI-1 plays a key role in radiation-induced EC death in the intestine and suggests that this contributes strongly to the progression of radiation-induced intestinal injury.

Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism.

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.