Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells

Objectives

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.

Results

Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.

Conclusion

Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

     

Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.     

Spatial and Temporal Localization of the Melanocortin 1 Receptor and Its Ligand ���CMelanocyte-Stimulating Hormone during Cutaneous Wound Repair

Growing evidence indicates that the melanocortin 1 receptor (MC1R) and its ligand α–melanocyte-stimulating hormone (α-MSH) have other functions in the skin in addition to pigment production. Activation of the MC1R/α-MSH signaling pathway has been implicated in the regulation of both inflammation and extracellular matrix homeostasis. However, little is known about the role of MC1R/α-MSH signaling in the regulation of inflammatory and fibroproliferative responses to cutaneous injury. Although MC1R and α-MSH localization has been described in uninjured skin, their spatial and temporal expression during cutaneous wound repair has not been investigated. In this study, the authors report the localization of MC1R and α-MSH in murine cutaneous wounds, human acute burns, and hypertrophic scars. During murine wound repair, MC1R and α-MSH were detected in inflammatory cells and suprabasal keratinocytes at the leading edge of the migrating epithelial tongue. MC1R and α-MSH protein levels were upregulated in human burn wounds and hypertrophic scars compared to uninjured human skin, where receptor and ligand were absent. In burn wounds and hypertrophic scars, MC1R and α-MSH localized to epidermal keratinocytes and dermal fibroblasts. This spatiotemporal localization of MC1R and α-MSH in cutaneous wounds warrants future investigation into the role of MC1R/α-MSH signaling in the inflammatory and fibroproliferative responses to cutaneous injury. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Divergent effects of canonical and non‐canonical TGF‐β signalling on mixed contractile‐synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.     

In vitro study of smooth muscle cells on polycaprolactone and collagen nanofibrous matrices

Biodegradable polycaprolactone and collagen nanofibers were produced by electrospinning, with fiber diameters of around 300-700nm and features similar to the extracellular matrix of natural tissue. Human coronary artery smooth muscle cells (SMCs) seeded on nanofibrous matrices tend to maintain normal phenotypic shape and growth tends to be guided by the nanofiber orientation. The SMC and nanofibrous matrix interaction was observed by SEM, MTS assay, trypan blue exclusion method and laser scanning confocal microscopy. The results showed that the proliferation and growth rate of SMCs were not different on polycaprolactone (PCL) nanofibrous matrices coated with collagen or tissue culture plates. PCL nanofibrous matrices coated with collagen showed that the SMCs migrated towards inside the nanofibrous matrices and formed smooth muscle tissue. This approach may be useful for engineering a variety of tissues in various structures and shapes, and also to demonstrate the importance of matching both the initial mechanical properties and degradation rate of nanofibrous matrices to the specific tissue engineering.