High Molecular Weight Caldesmon Research Articles

Transforming growth factor-beta 1 produced by vascular smooth muscle cells predicts fibrosis in the gastrocnemius of patients with peripheral artery disease.

BackgroundLower leg ischemia, myopathy, and limb dysfunction are distinguishing features of peripheral artery disease (PAD). The myopathy of PAD is characterized by myofiber degeneration in association with extracellular matrix expansion, and increased expression of transforming growth factor-beta 1 (TGF-β1; a pro-fibrotic cytokine). In this study, we evaluated cellular expression of TGF-β1 in gastrocnemius of control (CTRL) and PAD patients and its relationship to deposited collagen, fibroblast accumulation and limb hemodynamics.MethodsGastrocnemius biopsies were collected from PAD patients with claudication (PAD-II; N = 25) and tissue loss (PAD-IV; N = 20) and from CTRL patients (N = 20). TGF-β1 in slide-mounted specimens was labeled with fluorescent antibodies and analyzed by quantitative wide-field, fluorescence microscopy. We evaluated co-localization of TGF-β1 with vascular smooth muscle cells (SMC) (high molecular weight caldesmon), fibroblasts (TE-7 antigen), macrophages (CD163), T cells (CD3) and endothelial cells (CD31). Collagen was stained with Masson Trichrome and collagen density was determined by quantitative bright-field microscopy with multi-spectral imaging.ResultsCollagen density increased from CTRL to PAD-II to PAD-IV specimens (all differences p < 0.05) and was prominent around microvessels. TGF-β1 expression increased with advancing disease (all differences p < 0.05), correlated with collagen density across all specimens (r = 0.864; p < 0.001), associated with fibroblast accumulation, and was observed exclusively in SMC. TGF-β1 expression inversely correlated with ankle-brachial index across PAD patients (r = −0.698; p < 0.001).ConclusionsOur findings support a progressive fibrosis in the gastrocnemius of PAD patients that is caused by elevated TGF-β1 production in the SMC of microvessels in response to tissue hypoxia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0790-3) contains supplementary material, which is available to authorized users.

MiR-133 modulates TGF-β1-induced bladder smooth muscle cell hypertrophic and fibrotic response: Implication for a role of microRNA in bladder wall remodeling caused by bladder outlet obstruction

Bladder outlet obstruction (BOO) evokes urinary bladder wall remodeling significantly, including the phenotype shift of bladder smooth muscle cells (BSMCs) where transforming growth factor-beta1 (TGF-β1) plays a pivotal role given the emerging function of modulating cellular phenotype. miR-133 plays a role in cardiac and muscle remodeling, however, little is known about its roles in TGF-β1-induced BSMC hypertrophic and fibrotic response. Here, we verified BOO induced bladder wall remodeling and TGF-β1 expression mainly located in bladder endothelium. Furthermore, we uncovered miR-133a/b expression profile in BOO rats, and then explored its regulated effects on BSMCs' phenotypic shift. Our study found that miR-133 became down-regulated during rat bladder remodeling. Next, we sought to examine whether the expression of miR-133 was down-regulated in primary BSMCs in response to TGF-β1 stimulation and whether forced overexpression of miR-133 could regulate profibrotic TGF-β signaling. We found that stimulation of BSMCs with exogenous TGF-β1 of increasing concentrations resulted in a dose-dependent decrease of miR-133a/b levels and transfection with miR-133 mimics attenuated TGF-β1-induced α-smooth muscle actin, extracellular matrix subtypes and fibrotic growth factor expression, whereas it upregulated high molecular weight caldesmon expression compared with the negative control. Also, downregulation of p-Smad3, not p-Smad2 by miR-133 was detected. Additionally, miR-133 overexpression suppressed TGF-β1-induced BSMC hypertrophy and proliferation through influencing cell cycle distribution. Bioinformatics analyses predicted that connective tissue growth factor (CTGF) was the potential target of miR-133, and then binding to the 3′-untranslated region of CTGF was validated by luciferase reporter assay. These results reveal a novel regulator for miR-133 to modulate TGF-β1-induced BSMC phenotypic changes by targeting CTGF through the TGF-β–Smad3 signaling pathway. A novel antifibrotic functional role for miR-133 is presented which may represent a potential target for diagnostic and therapeutic strategies in bladder fibrosis.

Quantitative analysis of vessels with smooth muscle layer in astrocytic tumors: correlation with histological grade and prognostic significance.

Angiogenesis plays an important role in the progression of astrocytic tumors and its evaluation is a major prognostic factor. Although the form of proliferating vessels ranges from fine capillaries to well-developed vascular structures with a smooth muscle layer, the characteristics of vascular smooth muscle cells (SMCs) are not understood in detail. We therefore examined the density, size and shape of tumor vessels, as well as CD34-immunoreactive (CD34-Vs) or α-smooth muscle actin-immunoreactive (SMA-Vs) vessels in 46 primary astrocytomas (grade II diffuse astrocytomas, n=11, grade III anaplastic astrocytomas, n=15, grade IV glioblastomas, n=20) and in normal brain tissues from 10 autopsies. We also examined the expression of high molecular weight caldesmon (h-CD, a marker of the contractile phenotype of smooth muscle) and of platelet-derived growth factor receptor β (PDGFR-β). The SMA-Vs were significantly more dense and larger in grade IV than grade III, whereas those of CD34-Vs did not differ between grade III and IV. Changes in the shape of CD34-Vs and SMA-Vs correlated with histological grading. The expression of h-CD was reduced, whereas that of PFGFR-β was increased in high grade-astrocytomas. Kaplan-Meier analysis indicated that the density of SMA-Vs, the size of both CD34 and SMA-Vs and PDGFR-β expression were significant prognostic factors. These findings suggest that SMA-Vs are significantly associated with the progression of astrocytomas and that these vessels provide useful information for the histological diagnosis and survival of patients with these types of brain tumors.

Can lineage-specific markers be identified to characterize mesenchyme-derived cell populations in the human airways?

Mesenchyme-derived cells in the airway wall including airway smooth muscle cells, fibroblasts, and myofibroblasts are known to play important roles in airway remodeling. The lack of specific phenotypical markers makes it difficult to define these cell populations in primary cultures. Most relevant studies to date have used animal airway tissues, vascular tissues, or transformed cell lines with only limited studies attempting to phenotypically characterize human airway mesenchymal cells. The objectives of this study were to evaluate reported markers and identify novel markers to define these cell types. We could not identify any specific marker to define these cell populations in vitro that permitted unequivocal identification using immunocytochemistry. However, characteristic filamentous alpha-smooth muscle actin distribution was observed in a significant ( approximately 25%) proportion of human airway smooth muscle cells, whereas this was not observed in airway fibroblasts. A significantly higher proportion of airway fibroblasts expressed alpha(1)- and alpha(2)-integrin receptors compared with human airway smooth muscle cells as assessed by fluorescence activated cell sorting. Global gene expression profiling identified aldo-keto reductase 1C3 (AKR1C3) and cathepsin K as being novel markers to define airway smooth muscle cells, whereas integrin-alpha(8) (ITGA8) and thromboxane synthase 1 (TBXAS1) were identified as novel airway fibroblast-specific markers, and these findings were validated by RT-PCR. Ex vivo studies in human airway tissue sections identified high-molecular weight caldesmon and alpha-smooth muscle actin as being expressed in smooth muscle bundles, whereas ITGA8 and TBXAS1 were absent from these.

Pericryptal fibroblast sheath in intestinal metaplasia, dysplasia and carcinoma of the stomach

To investigate the existence of pericryptal fibroblasts sheath (PCFS) in normal gastric mucosa, intestinal metaplasia (IM), indefinite for dysplasia (I-Dys), low grade dysplasia (L-Dys), high grade dysplasia (H-Dys) and gastric cancer (GC), and its association with gastric carcinogenesis. In this study, we examined the existence of PCFS in normal gastric mucosa (N=10), IM (N=26), I-Dys (N=16), L-Dys (N=13), H-Dys (N=21) and GC (N=145) using immunohistochemical staining for two smooth muscle markers, alpha smooth muscle actin(α-SMA) and high molecular weight caldesmon (h-CD). The significance of PCFS was discussed, especially in association with gastric carcinogenesis. The PCFS was recognized in 65.4%(17/26) of IM, 62.5%(10/16) of I-Dys and 23.1% (3/13) of L-Dys respectively. No PCFS was detected in H-Dys and GC. The PCFS was gradually reduced in IM, Dys and GC in sequence (P<0.0001). The PCFS is associated with the differentiation of epithelium and involved in gastric carcinogenesis via epithelial-mesenchymal interaction.

Assessing the Effects of Transforming Growth Factor-β1 on Bladder Smooth Muscle Cell Phenotype. I. Modulation of In Vitro Contractility

Modulation of the bladder smooth muscle cell phenotype contributes to the resulting bladder dysfunction in many pathological bladder conditions. Transforming growth factor-beta1 is an important regulator of cellular phenotype in fibrotic diseases that has specific effects on bladder smooth muscle cells associated with phenotypic changes. We verified transforming growth factor-beta1 expression in neurogenic bladder tissue and investigated its effects on bladder smooth muscle cell collagen gel contraction. Transforming growth factor-beta1 immunostaining was performed on tissue sections from spinalized rats and quantified based on the ratio of fluorescence to total detrusor area. Rat bladder smooth muscle cells were seeded at different densities on anchored collagen gels and the effect of transforming growth factor-beta1 on contractility was assessed by measuring changes in the collagen gel area with time. Phenotypic changes induced by transforming growth factor-beta1 were detected by immunostaining for caldesmon and the specific isoform high molecular weight caldesmon. Transforming growth factor-beta1 immunostaining revealed increased levels specifically in the detrusor of spinal cord injured rats. Rat bladder smooth muscle cell contraction increased with larger cell populations and was inhibited by transforming growth factor-beta1. Transforming growth factor-beta1 induced a decrease in high molecular weight caldesmon expression in bladder smooth muscle cells. Increased transforming growth factor-beta1 expression in the detrusor of spinal cord injured rats implies up-regulation and localized signaling in response to injury. Bladder smooth muscle cells showed a loss of contractility in response to transforming growth factor-beta1 in all cell populations. A shift in phenotype was confirmed by high molecular weight caldesmon immunostaining. These results suggest that transforming growth factor-beta1 can modulate bladder smooth muscle cell function and may be a crucial regulator of bladder smooth muscle cell phenotype in pathological bladder conditions.

Distribution and role of myofibroblasts in human normal seminal vesicle stroma

We studied the distribution of myofibroblasts in the stroma of normal seminal vesicles. Twelve normal seminal vesicles obtained by surgery on the diagnosis of some diseases were selected, and we evaluated the distribution of myofibroblasts in the seminal vesicles using immunohistochemical and electron and immunoelectron microscopic techniques. Immunohistochemically, myofibroblasts, which were positive for alpha-smooth muscle actin (ASMA) and negative for high molecular weight caldesmon (h-CD), were observed in the stroma just beneath the epithelium of normal seminal vesicles. Moreover, an electron microscope examination revealed the presence of spindle or stellate cells in the stroma of the lamina propria beneath the seminal vesicle epithelium, and an immunoelectron microscopic examination showed that these cells were positive for ASMA. Finally, myofibroblasts are distributed in the lamina propria of human normal seminal vesicles and may play an important role in the ejection of sperm plasm.

Differential Expression of Smooth Muscle Myosin, Smooth Muscle Actin, H-Caldesmon, and Calponin in the Diagnosis of Myofibroblastic and Smooth Muscle Lesions of Skin and Soft Tissue

The diagnosis of low-grade and pseudosarcomatous spindle cell lesions of skin and soft tissue can sometimes be problematic; in particular, distinction between fibroblastic, myofibroblastic, and smooth muscle proliferations can occasionally pose difficulties on routine histologic examination. We have applied a panel of immunohistochemical markers to a series of spindle cell lesions of skin and soft tissue to assess the utility of the differential expression of smooth muscle and myofibroblastic-associated markers. Twenty-eight cases of nodular fasciitis, 42 cases of fibromatosis, and 3 cases of myofibroblastic sarcoma were stained with antibodies against smooth muscle actin (SMA), smooth muscle myosin (SMMS), calponin, and high-molecular weight caldesmon (h-caldesmon). For comparison, 12 cases of cutaneous leiomyoma and 8 cases of leiomyosarcomas involving superficial soft tissues and fascia were studied with the same panel of antibodies. Thirty-eight of 42 cases of fibromatosis were positive for SMA, 42/42 cases were positive for calponin, 39/42 cases were negative for SMMS, and all cases were negative for h-caldesmon. All cases of nodular fasciitis were positive for SMA and calponin, and all were negative for h-caldesmon and SMMS. All cases of myofibroblastic sarcoma were positive for SMA and 2/3 cases for calponin, and were negative for SMMS and h-caldesmon. All cases of cutaneous leiomyoma and leiomyosarcoma were positive for all 4 markers tested. Our results demonstrate a remarkably consistent pattern of reactivity of muscle and myofibroblastic-associated markers in lesions predominantly composed of myofibroblastic spindle cells, characterized by positive staining for SMA and calponin and absence of reactivity for SMMS and h-caldesmon. Application of this panel of stains may be of aid in the differential diagnosis of low-grade myofibroblastic lesions such as nodular fasciitis and fibromatosis from smooth muscle tumors of skin and soft tissue. This panel may additionally be of assistance in the diagnosis of myofibroblastic sarcoma.

Presence of vascular adventitial fibroblastic cells in diffuse-type gastric carcinomas

Aim: To investigate morphological changes in the tumour vessel adventitia, particularly the distribution of vascular adventitial fibroblastic cells (VAFCs)—namely, CD34 positive fibroblastic cells just outside the vascular media—in diffuse-type gastric...

Lack of vascular adventitial fibroblastic cells in tumour stroma of intestinal-type and solid-type gastric carcinomas

Aims: To investigate the roles of vascular adventitial fibroblastic cells in tumour stroma, the distribution of vascular adventitial fibroblastic cells was studied in gastric carcinomas. Methods: In total, 50 surgically...

High molecular weight caldesmon positive stromal cells in the capsule of thyroid follicular tumours and tumour-like lesions

Aims: To investigate the smooth muscle nature of the spindle stromal cells in the capsule of thyroid tumours and tumour-like lesions. Methods: Immunostaining for high molecular weight caldesmon (HCD), a...

α Smooth muscle actin positive stromal cells in gastric carcinoma

Aims: To investigate the distribution and roles of α smooth muscle actin (ASMA) positive stromal cells (ASMA+ cells), which belong to the myofibroblast group, within gastric carcinomas, with reference to...

CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells in the tumor capsule of skin sweat gland neoplasms.

To elucidate the roles of CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells at the tumor border of skin sweat gland neoplasms, we examined expression of stromal cell markers in the tumor capsule of 19 skin sweat gland neoplasms (16 mixed tumors of the skin and three nodular hidradenomas) using monoclonal antibodies to CD34, CD31, cytokeratin 14 (CK14), alpha-smooth muscle actin (ASMA) and high molecular weight caldesmon (HCD). We regarded CD34-positive, CD31-, CK14-, ASMA- and HCD-negative stromal cells to be CD34-positive stromal cells, and ASMA-positive, HCD-, CK14-, CD34- and CD31-negative stromal cells to be ASMA-positive stromal cells. CD34-positive stromal cells were detected in the tumor capsule of all 19 of the tumors examined. In nine of the 16 mixed tumors (56%) and all of the three nodular hidradenomas, ASMA-positive stromal cells were detected at the immediate inner side of the CD34-positive stromal cell layers. These results indicate that cellular components in the tumor capsules of mixed tumors of the skin and nodular hidradenomas are CD34-positive stromal cells and ASMA-positive stromal cells, and suggest that stromal cells of these two cell types are associated with tumor capsule formation of skin sweat gland neoplasms.

Fibronexus in "Malignant Fibrous Histiocytoma" of the Bone: A Case Report of Pleomorphic Myofibrosarcoma

The fibronexus (fibronexus junction) has been thought to be a characteristic ultrastructural feature of myofibroblasts, but it is controversial whether the fibronexus is also a characteristic of various myofibroblastic tumors. We report here a case of pleomorphic bone sarcoma (pleomorphic/storiform malignant fibrous histiocytoma) with fibronexus junctions arising in the head of the left humerus of a 70-year-old woman. By light microscopy the tumor was composed of large spindle or polygonal cells occasionally arranged in fascicles. Foamy giant cells with bizarre nuclei were not uncommon. Immunohistochemically, the tumor cells were positive diffusely for vimentin and focally for muscle actin ( HHF35 ) and alpha-smooth muscle actin but were negative for desmin, high-molecular weight caldesmon, and S-100 protein. Ultrastructurally, the tumor cells had indented nuclei and spindle or polygonal cytoplasm, with little rough endoplasmic reticulum and small vesicles, and rather numerous mitochondria. The tumor cells had myofilaments with focal densities in the periphery, fibronectin fibrils adjacent to intracellular myofilaments, and by definition, therefore, fibronexus junctions. These findings suggest myofibroblastic differentiation, and a diagnosis of pleomorphic myofibrosarcoma is thought to be appropriate. We believe that the fibronexus is a characteristic and useful ultrastructural feature for differentiating myofibroblastic tumors from other pleomorphic myogenic sarcomas.

Myofibrosarcoma of the bone: a clinicopathologic study.

Myofibroblastic tumors are fairly recently established soft tissue neoplasms. Although most of them appear to be benign, myofibrosarcoma of the soft tissue, seemingly their malignant counterpart, have been reported. We describe the clinicopathologic and radiologic features of four cases of myofibrosarcoma arising from the bone. All but one of the patients were women ranging in age from 60 to 71 years. Two tumors occurred in the metaphyses of distal femurs and the others arose in the iliac bones. On radiologic examination all tumors exhibited well-demarcated lytic destructive lesions without periosteal reaction. Two tumors were localized in the bone, whereas the other two extended into surrounding soft tissues. Histologically, all tumors were composed principally of a mixture of a cell-rich fascicular area and a hypocellular fibrous area. In the former area tumor cells had rather eosinophilic spindle-shaped wavy cytoplasm and were arranged in interlacing fascicles and small storiform patterns with variable numbers of inflammatory cells. Tumors occasionally showed prominent pleomorphism, and large cells with hyperchromatic nuclei were seen. In contrast, hypocellular areas had various features, including collagenous, hyalinous scar-like and rarely keloid-like areas. Focal coagulation necroses were present in all but one tumor. Immunohistochemically, the tumors were positive for vimentin, muscle actin (HHF35), alpha-smooth muscle actin, calponin, and desmin, whereas all of them were negative for high molecular weight caldesmon. On follow-up there was one fatal case with distant metastases, whereas the clinical courses of other cases after wide resection were excellent. Myofibrosarcoma of the bone has distinctive histopathologic features, which should be distinguished from those of other bone tumors with myoid differentiation.

Differential expression of high molecular weight caldesmon in colorectal pericryptal fibroblasts and tumour stroma.

AIM: To investigate an extent of smooth muscle differentiation of pericryptal fibroblasts. METHODS: Expression of high molecular weight caldesmon (h-CD) and alpha smooth muscle actin was investigated in 123 invasive...

Over‐expression of smooth muscle caldesmon in mouse fibroblasts

Caldesmon is an actin, calmodulin, tropomyosin, and myosin binding protein implicated in the regulation of actomyosin interactions. We have investigated the effect of overexpression of the higher molecular weight smooth muscle isoform of caldesmon on mouse L cell physiology. Mouse L(TK-) cells were transfected stably with plasmids carrying the TK+ gene and a full length human smooth muscle caldesmon cDNA under control of the adenovirus major late promoter. Two clones displaying four and eight times the level of the endogenous mouse high molecular weight caldesmon were isolated. These cells acquire a distinct phenotype characterized by an altered morphology, including an increased number of processes and larger area due to enhanced cell spreading, and a significantly slower growth rate than that of untransfected control cells, or cells transfected with the TK+ gene alone. The majority of the overexpressed caldesmon appears to be active and localized on cytoskeleton structures as determined by detergent lysis. Immunofluorescence analysis of the clones revealed that the caldesmon is localized as punctate staining on stress-fibers and in membrane ruffles. The immunofluorescence images suggest that caldesmon overexpressing cells have more total filaments than control cells. The effects of excess caldesmon on cell mobility are ambiguous: one clone displayed increased motility compared to the control, while the motility of the second clone was decreased relative to the control.

Genomic structure of the human caldesmon gene.

The high molecular weight caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for isoform diversity of human CaDs, l-CaD cDNAs were cloned from HeLa S3 cells. HeLa l-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa l-CaD I) are deleted in HeLa l-CaD II. The short amino-terminal sequence of HeLa l-CaDs is different from that of fibroblast (WI-38) l-CaD II and human aorta h-CaD. We have also identified WI-38 l-CaD I, which contains a 26-amino acid insertion relative to WI-38 l-CaD II. To reveal the molecular events of the expressional regulation of the CaD isoforms, the genomic structure of the human CaD gene was determined. The human CaD gene is composed of 14 exons and was mapped to a single locus, 7q33-q34. The 26-amino acid insertion is encoded in exon 4 and is specifically spliced in the mRNAs for both h-CaD and l-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaD (residues 73-207 for h-CaD and WI-38 l-CaDs, and residues 68-201 for HeLa l-CaDs). The regulation of h- and l-CaD expression is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change in expression between l-CaD and h-CaD might be caused by this splicing pathway.

Mapping of caldesmon: relationship between the high and low molecular weight forms

Caldesmon is a widely distributed contractile protein that occurs in both a high molecular weight [120-150-kilodalton (kDa)] and a low molecular weight (71-80-kDa) form, depending on the tissue. The structural relationship between these two forms was examined by mapping techniques. Partial cyanogen bromide cleavage in conjunction with sodium dodecyl sulfate gel electrophoresis was used to construct a map of the cleavage points and determine the relative position of the fragments in a high molecular weight caldesmon from chicken gizzard (caldesmon125). By use of this map, markers for different regions of the protein were obtained: Antibodies directed toward certain areas were prepared by affinity purification, and specific 125I-labeled tryptic peptides were found to originate from terminal cyanogen bromide fragments. Mapping of a lower molecular weight form of caldesmon (caldesmon72 from chicken liver) revealed the presence of sequences located in both ends of caldesmon125. A terminal 38-kDa fragment of both proteins was apparently identical on the basis of arrangement of cleavage sites, antibody reactivity, and iodopeptide mapping. Fragments from the other end of both proteins exhibited an identical pattern of peptides. These results show that it is sequences located in the central area of caldesmon125 which are missing in caldesmon72, indicating that the smaller molecule is not simply a proteolytic product of the larger. The two forms of caldesmon may be derived from separate genes or by alternative splicing from a single gene.

Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cells.

We investigated the expression of two molecular weight forms of caldesmon in a wide range of tissues and cells. The distribution of high molecular weight caldesmon (h-caldesmon, Mr 120,000-150,000) was restricted to smooth muscles where it was found in large quantity. The low molecular weight protein (l-caldesmon, Mr 70,000-80,000) was widely distributed in nonmuscle tissues and cells. Therefore, the expression of h-caldesmon might be much more specific to smooth muscles. We then examined the expressional changes of two caldesmons during phenotypic modulation of smooth muscle cells (SMCs). In developing gizzards, the expression of caldesmons switched from the l- to the h-form. Contrarily, the expression turned from h- to l-caldesmon in association with dedifferentiation of aortic SMCs in primary culture. In agreement with these observations, the levels of those mRNAs that direct the synthesis of both caldesmons were apparently in proportion to the quantities of protein, as determined by use of an in vitro translation system. In addition, h-caldesmon in smooth muscle-like BC3H1 cells increased in its amount with a concomitant reduction of l-caldesmon following serum-depleted and contact-inhibited cytodifferentiation. These results suggest that the expressional changes of two caldesmons are closely correlated with the phenotypic modulation of SMCs.