Abstract-We previously showed that changes in vascular smooth muscle cell (SMC) PTEN/Akt signaling following vascular injury are associated with increased SMC proliferation and neointima formation. In this report, we used a genetic model to deplete PTEN specifically in SMCs by crossing PTEN LoxP/LoxP mice to mice expressing Cre recombinase under the control of the SM22␣ promoter. PTEN was downregulated with increases in phosphorylated Akt in major vessels, hearts, and lungs of mutant mice. SMC PTEN depletion promoted widespread medial SMC hyperplasia, vascular remodeling, and histopathology consistent with pulmonary hypertension. Increased vascular deposition of the chemokine stromal cell-derived factor (SDF)-1␣ and medial and intimal cells coexpressing SM-␣-actin and CXCR4, the SDF-1␣ receptor, was detected in SMC PTEN-depleted mice. PTEN deficiency in cultured aortic SMCs induced autocrine growth through increased production of SDF-1␣. Blocking SDF-1␣ attenuated autocrine growth and blocked growth of control SMCs induced by conditioned media from PTEN-deficient SMCs. In addition, SMC PTEN deficiency enhanced progenitor cell migration toward SMCs through increased SDF-1␣ production. SDF-1␣ production by other cell types is regulated by the transcription factor hypoxia-inducible factor (HIF)-1␣. We found SMC nuclear HIF-1␣ expression in PTEN-depleted mice and increased nuclear HIF-1␣ in PTEN-deficient SMCs. Key Words: smooth muscle cell Ⅲ PTEN Ⅲ neointima Ⅲ autocrine growth Ⅲ conditional knockout mouse S mooth muscle cell (SMC) accumulation in the arterial intima is a key event in the pathogenesis of atherosclerosis, postangioplasty/in-stent restenosis, and graft arteriosclerosis, 1 with changes in the biological function and phenotype of SMCs contributing to the pathology. 2 These conditions are characterized, to varying degrees, by dedifferentiation, migration, and proliferation of medial-derived SMCs to form the neointima. Recent data suggest that bone marrow-derived, circulating, and/or resident progenitor/ proinflammatory cells are recruited to the injured vessel, differentiate down a SMC lineage, and proliferate, thereby contributing to neointimal lesion formation. [3][4][5][6] Compelling evidence supports the contribution of both processes to intimal hyperplasia, and major advances have identified numerous factors involved in this complex pathobiology. However, the underlying mechanism(s) initiating lesion formation are not clearly defined. Increased SMC production of chemokines, such as stromal cell-derived factor (SDF)-1␣ (CXCL12), has been shown to be centrally involved in progenitor cell recruitment, 7 although their role in inducing an autocrine growth pathway within the artery wall itself is unknown. We focused on the hypothesis that SMCs are central mediators of the injury response. Perturbations in SMC signaling, result in the production of soluble factors that regulate significant SMC hyperplasia and progenitor/proinflammatory cell recruitment through an autocrine/paracrine mechanism.Under physio...

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.