Mature Smooth Muscle Cells Research Articles

Differentiation of multipotent vascular stem cells contributes to vascular diseases

It is generally accepted that the de-differentiation of smooth muscle cells (SMCs) from contractile to proliferative/synthetic phenotype has an important role during vascular remodeling and diseases. Here we provide evidence that challenges this theory. We identify a new type of multipotent vascular stem cell (MVSC) in blood vessel wall. MVSCs express markers including Sox17, Sox10 and S100β, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell (MSC)-like cells that subsequently differentiate into SMCs. On the other hand, we use lineage tracing with smooth muscle myosin heavy chain as a marker to show that MVSCs and proliferative or synthetic SMCs do not arise from the de-differentiation of mature SMCs. Upon vascular injuries, MVSCs, instead of SMCs, become proliferative, and MVSCs can differentiate into SMCs and chondrogenic cells, thus contributing to vascular remodeling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of MVSCs, rather than the de-differentiation of SMCs, contributes to vascular remodeling and diseases.

Leiomyolipoma of the Orbit

Orbital leiomyolipoma is a rare benign tumor characterized by a mixture of mature smooth muscle cells and adipocytes. The authors present a case which, to the best of their knowledge, is the second case of leiomyolipoma of the orbit reported in the ophthalmic literature. A 65-year-old patient presented with a painful and swollen left eye, the symptoms having been present for 1 week. Imaging (CT, MRI follow up) confirmed the presence of a nearly 2-cm lesion medial to the lacrimal gland. A biopsy was planned, and 3 specimens were taken for histologic examination. Histology showed diffusely intermingling smooth muscle cells and mature adipocytes; immunohistochemistry demonstrated positive staining for smooth muscle actin and negativity for HMB45, MART-1, and Cytokeratin AE1/3. Although it is very rare, ophthalmologists should consider leiomyolipoma in the differential diagnosis of orbital tumors. The long-term prognosis for patients affected by this benign tumor is good.

Tumor vessel stabilization and remodeling by anti-angiogenic therapy with bevacizumab

Bevacizumab-resistant tumor vessels were characterized by an increased vessel diameter and normalization of vascular structures by the recruitment of mature pericytes and smooth muscle cells. Here, we analyzed human liver metastases which were taken at clinical relapse in patients with colorectal adenocarcinoma treated with anti-angiogenic therapy using the humanized monoclonal anti-VEGF bevacizumab. Tumor vessels which are resistant to anti-VEGF therapy are increased in size and characterized by a normalization of the vascular bed. These results were confirmed using NOD SCID mice as animal model and xenograft transplantation of human PC-3 prostate carcinoma cells in combination with bevacizumab treatment. Our results confirmed that anti-angiogenic therapy results in enhanced vascular remodeling by vascular stabilization. This process is apparently accompanied by enhanced necrosis of tumor tissue. These processes interfere with the efficacy of anti-angiogenic therapy because of reduced susceptibility of stabilized vessels by this therapy. These results demonstrate the importance for the development of second generation anti-angiogenic combination therapy concepts to rule out the balance between vascular stabilization followed by a possible de-stabilization making the remained vessels susceptible to a second wave of anti-angiogenic therapy.

The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells

BackgroundRecent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs. Methods and resultsUndifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase. ConclusionWe showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.

Derivation of multipotent nestin+/CD271−/STRO-1− mesenchymal-like precursors from human embryonic stem cells in chemically defined conditions

The successful establishment of stem cell-based therapies requires multipotent, immunocompatible stem cells, highly efficient strategies for direct differentiation, and most importantly, optimal culture conditions for large-scale expansion of such cell populations. Other than adult tissues, human embryonic stem cells (hESCs) represent another infinitely expansible source for mesenchymal stem cell (MSC) derivation. Here, we reproducibly derived a population of Nestin(+)/CD271(-)/STRO-1(-) mesenchymal-like precursors from hESCs (hESC-MPs) in chemically defined conditions, without requiring any serum or serum replacement of animal origin, based on a Y-27632-assisted monolayer culture system. These cells showed slim fibroblastic morphology, and satisfied the criteria of MSCs including self-renewal, the expression of multiple MSC-specific markers and the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Compared with previously reported hESC-derived MSCs, our hESC-MPs were more multipotent, and could differentiate into representative derivatives of all three embryonic germ layers including mature smooth muscle cells, cardiomyocytes, functional hepatocytes and neural cells expressing various neurotransmitter phenotypes, making them an attractive cell source for regenerative medicine.

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels. Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC). A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM). To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Pathophysiological features of intimal hyperplasia of the arterially implanted autovein graft and its anastomosis in dogs

We investigated pathophysiological features of intimal hyperplasia of arterially implanted autovein graft and its distal end-to-side anastomosis under conditions of poor distal runoff. Intimal hyperplasia of the graft was significantly evident, became 70.5±38.5 µm thick at 14 days with infiltration of myofibroblasts, and increased to 420±199.7 µm at 6 months. The proliferated neointima was strongly positive for alpha-smooth muscle (A-sm) actin staining. Cells in the entire layer of the graft were diffusely labeled with BrdU at 14 days. At 3–6 months after grafting, cells in the superficial layer of the neointima were scarcely labeled with BrdU, however, cells in the deeper layer were strongly labeled. At the distal end-to-side anastomosis, mural thrombi deposited on the suture line were replaced by fibrous tissues with infiltration of fibroblast-like cells within 14 days, and proliferated considerably at 3–6 months. The neointima at the toe portion was thickened to 82.5±50.7 µm at 14 days and increased to 370.6±40.0 µm at 6 months. The superficial layer of the proliferated neointima consisted of mature smooth muscle cells positive for A-sm actin staining and scarcely labeled with BrdU. However, the deeper layer was negative for A-sm actin staining and strongly labeled with BrdU. In conclusion, intimal hyperplasia is caused by infiltration of myofibroblasts in the graft and of fibroblast-like cells at the anastomosis. Proliferation of these cells is progressive at the deeper layer of the neointima, even at 6 months after grafting.

SDF-1α Induction in Mature Smooth Muscle Cells by Inactivation of PTEN Is a Critical Mediator of Exacerbated Injury-Induced Neointima Formation

PTEN inactivation selectively in smooth muscle cells (SMC) initiates multiple downstream events driving neointima formation, including SMC cytokine/chemokine production, in particular stromal cell-derived factor-1α (SDF-1α). We investigated the effects of SDF-1α on resident SMC and bone marrow-derived cells and in mediating neointima formation. Inducible, SMC-specific PTEN knockout mice (PTEN iKO) were bred to floxed-stop ROSA26-β-galactosidase (βGal) mice to fate-map mature SMC in response to injury; mice received wild-type green fluorescent protein-labeled bone marrow to track recruitment. Following wire-induced femoral artery injury, βGal(+) SMC accumulated in the intima and adventitia. Compared with wild-type, PTEN iKO mice exhibited massive neointima formation, increased replicating intimal and medial βGal(+)SMC, and enhanced vascular recruitment of bone marrow cells following injury. Inhibiting SDF-1α blocked these events and reversed enhanced neointima formation observed in PTEN iKO mice. Most recruited green fluorescent protein(+) cells stained positive for macrophage markers but not SMC markers. SMC-macrophage interactions resulted in a persistent SMC inflammatory phenotype that was dependent on SMC PTEN and SDF-1α expression. Resident SMC play a multifaceted role in neointima formation by contributing the majority of neointimal cells, regulating recruitment of inflammatory cells, and contributing to adventitial remodeling. The SMC PTEN-SDF-1α axis is a critical regulator of these events.

Human Coronary Artery Smooth Muscle Cell Responses to Bioactive Polyelectrolyte Multilayer Interfaces

Under normal physiological conditions, mature human coronary artery smooth muscle cells (hCASMCs) exhibit a “contractile” phenotype marked by low rates of proliferation and protein synthesis, but these cells possess the remarkable ability to dedifferentiate into a “synthetic” phenotype when stimulated by conditions of pathologic stress. A variety of polyelectrolyte multilayer (PEMU) films are shown here to exhibit bioactive properties that induce distinct responses from cultured hCASMCs. Surfaces terminated with Nafion or poly(styrenesulfonic acid) (PSS) induce changes in the expression and organization of intracellular proteins, while a hydrophilic, zwitterionic copolymer of acrylic acid and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-PAEDAPS) is resistant to cell attachment and suppresses the formation of key cytoskeletal components. Differential expression of heat shock protein 90 and actin is observed, in terms of both their magnitude and cellular localization, and distinct cytoplasmic patterns of vimentin are seen. The ionophore A23187 induces contraction in confluent hCASMC cultures on Nafion-terminated surfaces. These results demonstrate that PEMU coatings exert direct effects on the cytoskeletal organization of attaching hCASMCs, impeding growth in some cases, inducing changes consistent with phenotypic modulation in others, and suggesting potential utility for PEMU surfaces as a coating for coronary artery stents and other implantable medical devices.

A Differentiation-Based MicroRNA Signature Identifies Leiomyosarcoma as a Mesenchymal Stem Cell-Related Malignancy

Smooth muscle (SM) is a spontaneously contractile tissue that provides physical support and function to organs such as the uterus. Uterine smooth muscle-related neoplasia comprise common well-differentiated benign lesions called leiomyomas (ULM), and rare, highly aggressive and pleomorphic tumors named leiomyosarcomas (ULMS). MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in normal cellular development and tissue homeostasis that can be used to accurately subclassify different tumor types. Here, we demonstrate that miRNAs are required for full smooth muscle cell (SMC) differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). We also report a miRNA signature associated with this process. Moreover, we show that this signature, along with miRNA profiles for ULMS and ULM, are able to subclassify tumors of smooth muscle origin along SM differentiation. Using multiple computational analyses, we determined that ULMS are more similar to hMSCs as opposed to ULM, which are linked with more mature SMCs and myometrium. Furthermore, a comparison of the SM differentiation and ULMS miRNA signatures identified miRNAs strictly associated with SM maturation or transformation, as well as those modulated in both processes indicating a possible dual role. These results support separate origins and/or divergent transformation pathways for ULM and ULMS, resulting in drastically different states of differentiation. In summary, this work expands on our knowledge of the regulation of SM differentiation and sarcoma pathogenesis.

Foetal and adult cardiomyocyte progenitor cells have different developmental potential

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. Up to date, no studies have been reported in which the developmental potential of foetal and adult cardiovascular progenitors was tested simultaneously. However, intrinsic differences will likely affect interpretations regarding progenitor cell potential and application for regenerative medicine. Here we report a direct comparison between human foetal and adult heart-derived cardiomyocyte progenitor cells (CMPCs). We show that foetal and adult CMPCs have distinct preferences to differentiate into mesodermal lineages. Under pro-angiogenic conditions, foetal CMPCs form more endothelial but less smooth muscle cells than adult CMPCs. Foetal CMPCs can also develop towards adipocytes, whereas neither foetal nor adult CMPCs show significant osteogenic differentiation. Interestingly, although both cell types differentiate into heart muscle cells, adult CMPCs give rise to electrophysiologically more mature cardiomyocytes than foetal CMPCs. Taken together, foetal CMPCs are suitable for molecular cell biology and developmental studies. The potential of adult CMPCs to form mature cardiomyocytes and smooth muscle cells may be essential for cardiac repair after transplantation into the injured heart.

In-Vivo Autologus Bladder Wall Generation; Application of Different Scaffolds for Bladder Autoaugmentation in a Model of Bladder as a Bioreactor

Purpose To investigate the potential of different scaffolds in bladder wall construction, using bladder as a bioreactor. Material and Methods Forty rabbits were divided into 10 groups. The control group underwent classical autoaugmentation (G1). In all other groups, different scaffolds were implanted between bladder mucosa and seromuscular layer. A biopsy of 10x5 mm2 full thickness of bladder was dissected. The smooth muscle cell (SMC) and urothelial cell (UC) layers were separated and minced into 20 fragments. The SMC fragments were seeded on mucosal layer and UC fragments were placed on 2x1cm2 framed scaffold under seromuscular layer.The scaffolds used in each groups were as follow: G2:acellular pericardium, G3:biofilm, G4:Polyglycolic Acid (PGA) as a biodegradable scaffold, G5:biofilm-coated acellular pericardium, G6:PGA-coated pericardium, G7:Pericardium-coated Biofilm, G8:PGA-coated Biofilm, G9:Pericarduim-coated PGA, and G10:Biofilm-coated PGA. After 2 and 6 weeks, biopsies were performed for histologic examinations and then the recombinant bladder tissue layers were grafted to the remaining host bladder. Biopsies at 1month intervals were obtained for determination of CD31/34, SMC α-actin, and cytokeratin AE1/AE3, following cystometry. Results Histopathological examinations revealed granulation in G3, 5, 7, 8, and 10.Bladder wall frames in G2 and 6 demonstrated organized bladder wall generation in two different expanded layers with mature UCs and SMCs and showed a significant bladder capacity increment as compared with other groups(P Conclusions Our results demonstrated effective role of tissue-engineered pericardium as a potential scaffold for SMC and UC seeding in bladder wall acting as a natural bioreactor. The biodegradable scaffolds could be also helpful in association with acellular matrixes in improvement of cell attachment and optimizing the in-vivo bladder wall construction for bladder auto augmentation.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

Lipoleiomyoma of uterus in a postmenopausal woman

Lipoleiomyomas are uncommon benign neoplasms of uterus and are considered to be a variant of uterine myomas. Their reported incidence varies from 0.03 to 0.2%. Lipoleiomyoma consists of variable proportion of mature lipocytes and smooth muscle cells. These tumors generally occur in asymptomatic obese perimenopausal or menopausal women. We report this case of uterine lipoleiomyoma because of its rarity.

Sex steroid-mediated reprogramming of vascular smooth muscle cells to stem cells and neurons: Possible utilization of sex steroid combinations for regenerative treatment without utilization of in vitro developed stem cells

Previous work from our laboratory demonstrated that sex steroid combinations, but not individual sex steroids alone, cause transdifferentiation of ovarian epithelial cells - ovarian surface epithelium (OSE) and follicular granulosa cells - into neural stem cells (NSC) and differentiating neurons. In the present study we have chosen primary culture of human vascular smooth muscle cells (SMC), a non-epithelial mesenchymal cells in order to test them as a control cell type regarding their morphology and expression of NSC and neuronal markers. Utilization of estradiol (E2), progesterone (PG) or testosterone (TS) alone did not induce the emergence of neurons from the vascular SMC. However, the treatment with sex steroid combinations (PG+TS or E2+PG+TS) caused transdifferentiation into neural/neuronal type cells. By immunohistochemistry, these cells exhibited strong expression of stem cell markers and neural/neuronal glycoconjugates SSEA-1, SSEA-4, Thy-1, NeuN and NCAM. In the Neurobasal/B27 medium both, the OSE and vascular SMC also transdifferentiated into neuronal cells. Western blot analysis has shown significant increase of NeuN 48-kDa species after E2+PG or PG+TS treatment. Secretion of E2 increased significantly in vascular SMC cultures pretreated with TS, PG or TS+PG. Unlike OSE cells, the vascular SMC accompany as pericytes all vessels, including CNS microvasculature. We also observed that sex steroid combinations could produce SMC stem type cells which differentiated within a few days back to mature vascular SMC. This is of potential interest for the vascular regenerative medicine. Altogether, our observations suggest that sex steroid combinations could induce in vivo improvement of neurodegenerative, traumatic and ischemic neurological disorders and vascular diseases via their effect on resident pluripotent vascular SMC, i.e. without a need of in vitro developed stem cells.

A Specific Subset of Mouse Bone Marrow Cells Has Smooth Muscle Cell Differentiation Capacity–Brief Report

To determine whether CX(3)CR1(+) bone marrow cells have the capacity for smooth muscle cell (SMC) differentiation. CX(3)CR1(+) and CX(3)CR1(-) cells were isolated from marrow of CX(3)CR1 transgenic mice and cultured in SMC differentiation media. Phenotypic and functional analyses showed only CX(3)CR1(+) bone marrow cells exhibit colony cell outgrowth with SMC-specific protein expression, calcium signaling, and contraction responses similar to mature SMC. CX(3)CR1 marks a bone marrow cells population that enriches for progenitors with capacity to differentiate in vitro into SMC-like cells.

Abstract 5431: Activation of Myocardin Function by an Ubiquitin E3 Ligase, EDD

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of contractile proteins. The expression of these proteins is markedly attenuated during the de-differentiation of smooth muscle that occurs under many pathological conditions. SRF is a transcription factor that plays a central role in the expression of these smooth muscle-specific genes through physical association with various cell-restricted or signal dependent accessory factors. Of the SRF-associated proteins identified, myocardin is the most potent for stimulating expression of smooth muscle-specific genes. Although several proteins have been identified that can modify myocardin function, the mechanism of how myocardin regulation is still poorly understood. To identify additional myocardin associated proteins that are required for myocardin-dependent activation smooth muscle-specific genes we performed a yeast 2-hybrid screen of mouse embryo cDNA library using myocardin as bait. From this screen we identified a HECT domain containing protein EDD as a myocardin binding protein. Previous studies have shown that HECT domain containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. EDD has however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that the amino-terminal domain of myocardin bound to the HECT domain of EDD. We also show that EDD specifically enhanced trans-activation of smooth muscle-specific promoters by myocardin. Furthermore, EDD significantly augmented myocardin’s ability to induce expression of endogenous SMC marker genes, and these effects of EDD were independent on its E3 ligase function. Conversely, depletion of endogenous EDD in fibroblast cells by RNAi attenuated myocardin-induced smooth muscle-specific gene expression, and EDD knockdown in smooth muscle cells resulted in down-regulation of smooth muscle-specific genes. Ongoing experiments are investigating the mechanism by which EDD activates myocardin function. This study reveals an unexpected role for the ubiquitin E3 ligase EDD as an activator of smooth muscle differentiation through its ability to potentiate myocardin function. This research has received full or partial funding support from the American Heart Association, National Center.

MP-17.05: Comparing the Success Rate of Mature Human Bladder Smooth Muscle Cells Culture on Two Different Kinds of Matrix: Human Amniotic Membrane and Collagen

Our goal in the present study is evaluating and comparing the natural behaviors, growth pattern, morphology and specific features of human bladder smooth muscle cells (HBSMCs) on two different matrix including human amniotic membrane (HAM) and collagen.

Expression of CPI-17 in smooth muscle during embryonic development and in neointimal lesion formation.

Ca(2+) sensitivity of smooth muscle (SM) contraction is determined by CPI-17, an inhibitor protein for myosin light chain phosphatase (MLCP). CPI-17 is highly expressed in mature SM cells, but the expression level varies under pathological conditions. Here, we determined the expression of CPI-17 in embryonic SM tissues and arterial neointimal lesions using immunohistochemistry. As seen in adult animals, the predominant expression of CPI-17 was detected at SM tissues on mouse embryonic sections, whereas MLCP was ubiquitously expressed. Compared with SM alpha-actin, CPI-17 expression doubled in arterial SM from embryonic day E10 to E14. Like SM alpha-actin and other SM marker proteins, CPI-17 was expressed in embryonic heart, and the expression was down-regulated at E17. In adult rat, CPI-17 expression level was reduced to 30% in the neointima of injured rat aorta, compared with the SM layers, whereas the expression of MLCP was unchanged in both regions. Unlike other SM proteins, CPI-17 was detected at non-SM organs in the mouse embryo, such as embryonic neurons and epithelium. Thus, CPI-17 expression is reversibly controlled in response to the phenotype transition of SM cells that restricts the signal to differentiated SM cells and particular cell types.

Lentiviral tracking of vascular differentiation in bone marrow progenitor cells

Lentiviral vectors encoding for identifiable marker genes controlled by lineage-specific promoters can be used to track differentiation of bone marrow progenitors into endothelial cells and/or smooth muscle cells. Human VE-Cadherin and Smoothelin-B promoters were cloned into a self-inactivating lentiviral vector (HR-VECad and HR-SMTHB) and used to drive expression of green fluorescent protein (eGFP). These constructs demonstrated specific promoter activity in mature endothelial and smooth muscle cells respectively in vitro. Lin − bone marrow progenitor cells (Lin − BMCs) in culture were used to test vector ability to track vascular differentiation. HR-VECad transduced Lin − BMCs were plated on collagen-coated slides and grown in endothelial media, while HR-SMTHB transduced Lin − BMCs were cultured on fibronectin-coated slides and grown in smooth muscle media. For in vivo differentiation assessment, lentiviral transduced Lin − BMCs resuspended in Matrigel were injected subcutaneously into C57BL/6J mice. Explants were evaluated for eGFP expression. Lin − BMCs grown in endothelial differentiation media produced groups of polygonal endothelial-like cells by days 16–21. When transduced with HR-VECad vector, these expressed eGFP in distinct cells within the colony by days 18–21, and coexpressed VE-Cadherin and eNOS. Lin − BMCs grown in smooth muscle differentiation media produced spindle-shaped cells between days 10–14 in culture. When transduced with the HR-SMTHB vector, these cells showed eGFP expression at ∼12 days, which increased over time and coexpressed αSMA, calponin and myosin heavy chain. Within Matrigel plugs containing HR-VECad transduced cells, eGFP + constituted 0.4±0.2% of total cells. In contrast, within Matrigel plugs containing HR-SMTHB transduced cells, eGFP + cells constituted 0.2±0.1% of total cells. These data demonstrate the feasibility of selectively marking BMC populations for cell fate determination.