Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.

Abstract-The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation.Smooth muscle expresses two isoforms of cGKI, cGKI␣ and cGKI␤, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca 2ϩ concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKI␣ and cGKI␤. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca 2ϩ transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKI␣ and cGKI␤. The defective Ca 2ϩ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKI␣ and enhanced green fluorescent protein (EGFP) but not by a cGKI␤-EGFP construct. Fluorescence imaging indicated that the cGKI␣-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKI␤-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKI␣ isoform, which decreases the noradrenaline-stimulated cytosolic Ca T he mechanism by which NO and NO-generating drugs lead to vasodilatation is not well understood. 1-5 Recent studies with knockout mice have shown that NO can induce the relaxation of vascular smooth muscle by activation of soluble guanylyl cyclase, production of cGMP, and activation of cGMP-dependent protein kinase I (cGKI). 6,7 Furthermore, NO and cGMP may regulate vascular tone by signaling pathways that do not require cGKI. 7,8 The identity of the cellular substrates and mechanisms regulated by cGKI has been discussed controversially. cGKI-mediated smooth muscle relaxation may involve a decrease in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) 6,9,10 as well as a modulation of the activity of myosin phosphatase, 11-14 RhoA, 15 or telokin. 16 Overexpression of cGKI in non-smooth muscle cells suggests additional targets. 1,17 At present, the functional significance of these targets in the regulation of vascular tone is unclear. Moreover, the interpretation of some results obtained with intact vascular smooth muscle cells (VSMCs) may be limited by the fact that the extensively used "specific cGKI inhibitor" KT5823 may not inhibit cGKI activity in intact cells 18 and that cGKI expression decreases during cell culture. 9,19 The cGKI gene encodes two isoforms, cGKI␣ and cGKI␤, which differ in their amino termini (Ϸ100 amino acids). 20,21 Both isoforms are expressed in vascular and nonvascular smooth muscle. [22][23][24] The amino terminus is involved in homodimerization, regulation of cGMP affinity and kinase activation, and target protein recognition. 1,2,25 Recent experiments have shown that the amino terminus of cGKI␣ interacts specifically with the m...

Nitric oxide (NO) exerts both antiatherogenic and proatherogenic effects, but the cellular and molecular mechanisms that contribute to modulation of atherosclerosis by NO are not understood completely. The cGMP-dependent protein kinase I (cGKI) is a potential mediator of NO signaling in vascular smooth muscle cells (SMCs). Postnatal ablation of cGKI selectively in the SMCs of mice reduced atherosclerotic lesion area, demonstrating that smooth muscle cGKI promotes atherogenesis. Cell-fate mapping indicated that cGKI is involved in the development of SMC-derived plaque cells. Activation of endogenous cGKI in primary aortic SMCs resulted in cells with increased levels of proliferation; increased levels of vascular cell adhesion molecule-1, peroxisome proliferator-activated receptor ␥, and phosphatidylinositol 3-kinase͞Akt signaling; and decreased plasminogen activator inhibitor 1 mRNA, which all are potentially proatherogenic properties. Taken together, these results highlight the pathophysiologic significance of vascular SMCs in atherogenesis and identify a key role for cGKI in the development of atherogenic SMCs in vitro and in vivo. We suggest that activation of smooth muscle cGKI contributes to the proatherogenic effect of NO and that inhibition of cGKI might be a therapeutic option for treating atherosclerosis in humans.A therosclerosis causes heart attack and stroke, the major causes of death in industrial nations. The pathophysiology of atherogenesis is complex. It is considered to be a chronic inflammatory condition that results from the interaction between modified lipoproteins and various cell types, including leukocytes, platelets, and cells of the vessel wall (1). The development of an atherosclerotic plaque involves, in addition to inflammation, the phenotypic modulation of vascular smooth muscle cells (SMCs) to proliferating and dedifferentiated cells. However, the mechanisms that contribute to the development of plaque SMCs and their pathophysiologic significance are not well understood (2).The signaling molecule nitric oxide (NO) has critical roles in the pathogenesis of atherosclerosis (3). Analysis of transgenic mice that lack or overexpress NO synthases indicated that NO exerts both protective (4, 5) and atherogenic (6-9) effects. The double role of NO might explain why NO-generating drugs (e.g., glyceryl trinitrate) have not been reported to limit the progression of atherosclerosis in humans. The opposing actions of NO on atherogenesis might depend on the spatiotemporal profile of its production and are likely mediated by different cellular and molecular mechanisms. Signaling pathways in SMCs that contribute to NO modulation of atherogenesis have not been identified. In vascular SMCs, NO is thought to exert many of its effects by activation of soluble guanylyl cyclase, synthesis of the second messenger cGMP, and activation of cGMP-dependent protein kinase I (cGKI) (10). The analysis of the functional significance of cGKI in NO͞cGMP signaling is complicated by the existence of multiple receptors ...

No abstract

No abstract