ABSTRACT
OBJECTIVES: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. APPROACH AND RESULTS: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. CONCLUSIONS: The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.
Subject(s)
Cardiovascular Diseases/drug therapy , Drug Evaluation, Preclinical/methods , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Lipoproteins, LDL/drug effects , Animals , Cardiovascular Diseases/blood , Cells, Cultured , Endothelial Cells/drug effects , Humans , In Vitro Techniques , Lipoproteins, LDL/metabolism , Macaca fascicularis , Models, Cardiovascular , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Species SpecificityABSTRACT
OBJECTIVE: Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. METHODS AND RESULTS: Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. CONCLUSIONS: These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.
Subject(s)
Aortic Aneurysm, Abdominal/metabolism , Apoptosis , Carotid Artery Diseases/metabolism , Cyclin-Dependent Kinase Inhibitor p15/deficiency , Muscle, Smooth, Vascular/metabolism , Tumor Suppressor Protein p53/metabolism , Adolescent , Adult , Aged , Animals , Aorta, Abdominal/metabolism , Aorta, Abdominal/pathology , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/pathology , Aortic Aneurysm, Abdominal/prevention & control , Apoptosis/drug effects , Benzothiazoles/pharmacology , Bone Marrow Transplantation , Carotid Arteries/metabolism , Carotid Arteries/pathology , Carotid Artery Diseases/genetics , Carotid Artery Diseases/pathology , Carotid Artery Diseases/prevention & control , Case-Control Studies , Cell Movement , Cell Proliferation , Cells, Cultured , Child , Child, Preschool , Cyclin-Dependent Kinase Inhibitor p15/genetics , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Disease Models, Animal , Gene Expression Regulation , Genotype , Humans , Infant , Infant, Newborn , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Neointima , Pancreatic Elastase , Phenotype , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , Signal Transduction , Time Factors , Toluene/analogs & derivatives , Toluene/pharmacology , Transfection , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.
