Skin-derived precursors from human subjects with Type 2 diabetes yield dysfunctional vascular smooth muscle cells.

Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, ∼1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7 ± 13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was ∼50% lower than those without T2D (cells/g: 0.18 ± 0.03, N=58 versus 0.40 ± 0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,207 ± 24,669, N=7 versus 101,537 ± 15,881, N=20, P<0.08) despite a reduced frequency of Ca2+ cycling (events s-1 cell-1: 0.011 ± 0.004, N=8 versus 0.021 ± 0.003, N=19, P<0.05) than those without T2D. SKP-derived VSMCs from subjects with T2D also manifest enhanced sensitivity to phenylephrine (PE) in an impedance-based assay (EC50 nM: 72.3 ± 63.6, N=5 versus 3,684 ± 3,122, N=9, P<0.05), and impaired wound closure in vitro (% closure: 21.9 ± 3.6, N=4 versus 67.0 ± 10.3, N=4, P<0.05). Compared with aortic- and saphenous vein-derived primary VSMCs, SKP-derived VSMCs are functionally distinct, but mirror defects of T2D also exhibited by primary VSMCs. CONCLUSION: Skin biopsies from older adults yield sufficient SKPs to differentiate VSMCs, which reveal abnormal phenotypes of T2D that survive differentiation and persist even after long-term normoglycemic culture.

Vascular smooth muscle cell differentiation from human stem/progenitor cells.

Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promote angiogenesis and wound healing. However, VSMCs are derived from diverse embryonic sources which may influence their role in the development of vascular disease and in its therapeutic modulation. Despite progress in understanding the mechanisms of VSMC differentiation, there remains a shortage of robust methods for generating lineage-specific VSMCs from pluripotent and adult stem/progenitor cells in serum-free conditions. Here we describe a method for differentiating pluripotent stem cells, such as embryonic and induced pluripotent stem cells, as well as skin-derived precursors, into lateral plate-derived VSMCs including 'coronary-like' VSMCs and neural crest-derived VSMC, respectively. We believe this approach will have broad applications in modeling origin-specific disease vulnerability and in developing personalized cell-based vascular grafts for regenerative medicine.

Peptide-mediated disruption of calmodulin-cyclin E interactions inhibits proliferation of vascular smooth muscle cells and neointima formation.

RATIONALE: Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. OBJECTIVE: Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca(2+)-sensitive G(1)-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. METHODS AND RESULTS: A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca(2+)-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G(1)-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited (3)H-thymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22α expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. CONCLUSIONS: A synthetic peptide designed to disrupt CaM-cyclin E binding inhibits Ca(2+)/CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis.

Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells.

OBJECTIVE: The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2(+) multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. METHODS AND RESULTS: We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-ß1 or -ß3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle γ-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. CONCLUSIONS: SKPs are Sox2(+) progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization.

Aortic Sca-1+ Progenitor Cells Arise from the Somitic Mesoderm Lineage in Mice.

Sca-1+ progenitor cells in the adult mouse aorta are known to generate vascular smooth muscle cells (VSMCs), but their embryological origins and temporal abundance are not known. Using tamoxifen-inducible Myf5-CreER mice, we demonstrate that Sca-1+ adult aortic cells arise from the somitic mesoderm beginning at E8.5 and continue throughout somitogenesis. Myf5 lineage-derived Sca-1+ cells greatly expand in situ, starting at 4 weeks of age, and become a major source of aortic Sca-1+ cells by 6 weeks of age. Myf5-derived adult aortic cells are capable of forming multicellular sphere-like structures in vitro and express the pluripotency marker Sox2. Exposure to transforming growth factor-ß3 induces these spheres to differentiate into calponin-expressing VSMCs. Pulse-chase experiments using tamoxifen-inducible Sox2-CreERT2 mice at 8 weeks of age demonstrate that ∼35% of all adult aortic Sca-1+ cells are derived from Sox2+ cells. The present study demonstrates that aortic Sca-1+ progenitor cells are derived from the somitic mesoderm formed at the earliest stages of somitogenesis and from Sox2-expressing progenitors in adult mice.

Enhanced Cardiac Differentiation of Human Cardiovascular Disease Patient-Specific Induced Pluripotent Stem Cells by Applying Unidirectional Electrical Pulses Using Aligned Electroactive Nanofibrous Scaffolds.

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (ß) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.

Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells.

AIMS: Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. METHODS AND RESULTS: Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo. CONCLUSION: The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.

Essential role for Schizosaccharomyces pombe pik1 in septation.

BACKGROUND: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4(G107S). PRINCIPAL FINDINGS: Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25 degrees C. At 36 degrees C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1(D709A) was kinase-dead, but bound Cdc4. Pik1(R838A) did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1(D709A,R838A) was innocuous, pik1(R838A) was almost innocuous, and pik1(D709A) produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4(G107S). Thus, D709 is essential for kinase activity and septation. CONCLUSIONS: Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.

Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis.

Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III beta four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.