Victor K. Lin

Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD

In this report, we describe the isolation, sequence, and initial characterization of the cDNA for the muscle-specific regulatory factor skeletal myogenin. Transfection of myogenin into the mesenchymal cell line C3H10T1/2 produces cells expressing muscle-specific markers. Myogenin is absent in undifferentiated cells, peaks, and then declines following a stimulus to differentiate, and is overexpressed in myoblasts selected with 5-bromodeoxyuridine for the overproduction of factors that regulate the decision to differentiate. High levels of myogenin transcripts are present in the myotomal region of somites at 8.5 days of gestation in the mouse. Although myogenin and MyoD are different genes, they share the myc homology domain. Myogenin and MyoD thus form part of a gene family regulating myogenesis, and together with myd may constitute a set of factors that interact to regulate the determination and differentiation of muscle cells.

CONTRACTILE PROTEIN EXPRESSION IN BLADDER SMOOTH MUSCLE IS A MARKER OF PHENOTYPIC MODULATION AFTER OUTLET OBSTRUCTION IN THE RABBIT MODEL

PURPOSE We determined changes in contractile protein expression before and after the relief of partial bladder outlet obstruction in the rabbit model and assessed their potential role as predictors of recovery. MATERIALS AND METHODS We examined the ratio of the smooth muscle myosin heavy chain isoforms SM2-to-SM1, caldesmon isoform expression and bladder function in obstructed and unobstructed adult rabbit bladders. Cystometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were done to determine changes in bladder function and contractile protein expression. RESULTS Overall we observed significant correlation of bladder weight with the SM2-to-SM1 ratio (p <0.05). Regardless of the duration of obstruction (up to 10 weeks) the ratio appeared to stabilize around a value comparable to that in fetal rabbit smooth muscle cells, suggesting a reversal of SM2 and SM1 expression to a level similar to that at the fetal stage. The pattern of h and l-caldesmon isoform expression showed an increase in l-caldesmon expression in obstructed bladders. Except for decreased leak point pressure in the obstructed group we noted no statistically significant urodynamic changes in bladder capacity or compliance. CONCLUSIONS There is significant correlation of bladder weight, which is the best known marker of obstruction, with the SM2-to-SM1 ratio. The myosin heavy chain isoform expression ratio appears to be an indicator of phenotypic modulation in bladder smooth muscle before and after the relief of bladder outlet obstruction. Thus, it may be useful as a marker of bladder dysfunction and predictor of functional recovery. Regression to a fetal pattern of protein expression may suggest irreversible damage to smooth muscle cells, possibly limiting recovery.

Elastin expression and elastic fibre width in the anterior vaginal wall of postmenopausal women with and without prolapse.

To compare elastin expression and elastic fibre width in the anterior vaginal wall of postmenopausal women with and with no bladder prolapse.

Myosin heavy chain gene expression in normal and hyperplastic human prostate tissue

Benign prostatic hyperplasia (BPH) is common among aging men. Over 80% of males 50–60 years and older have various degrees of bladder outlet obstruction secondary to BPH. Despite the tremendous medical impact of BPH, its molecular pathophysiology remains unclear. Current BPH research focuses on steroid hormonal effects, stromal‐epithelial cell interaction, and oncogenes and growth factors. But little is known about the potential prostatic smooth muscle (SM) alterations that may occur during stromal hyperplasia.

Extracellular matrix expression of human prolapsed vaginal wall

To compare the mRNA expression of extracellular matrix (ECM) proteins in postmenopausal prolapsed versus non‐prolapsed anterior vaginal wall (AVW) tissue. We hypothesized that the weakening of the tissue leading to prolapse was due to decreased collagen production from a downregulation at the transcriptional level.

Alpha-blockade downregulates myosin heavy chain gene expression in human benign prostatic hyperplasia

OBJECTIVES Smooth muscle (SM), a major component of prostate stroma, plays an important role in the pathogenesis of benign prostatic hyperplasia. In many muscle systems, steroid hormones and alpha(1)-adrenergic neurotransmitters tightly regulate expression of contractile proteins. In this study, SM content and the expression of myosin heavy chain (MHC) in tissues from patients with benign prostatic hyperplasia treated with androgen ablation or alpha-blockade were compared with untreated controls. METHODS Prostatic periurethral tissue specimens from patients receiving luteinizing hormone-releasing hormone analogues (n = 12), alpha-blocking agents (n = 12), and no treatment (n = 13) were examined. The samples were analyzed for SM MHC mRNA expression using competitive reverse transcription-polymerase chain reaction. SM content was measured by morphometric analysis of trichrome-stained sections. RESULTS Stromal SM constituted 45.4% +/- 8.6%, 48.1% +/- 18.4%, and 45.9% +/- 10.8% of the total tissue in androgen ablated, alpha-blocked, and untreated tissues, respectively. No significant difference was observed among these three groups (P = 0.84, analysis of variance). However, SM MHC mRNA expression was markedly decreased in the alpha-blockade group (0.15 +/- 0.02 attomole/mg tissue) compared with the androgen-ablated (0.58 +/- 0.15 attomole/mg tissue) or control (0.44 +/- 0.10 attomole/mg tissue) groups. The relationship between SM content and expression of SM MHC significantly differed among the groups (P = 0.02, analysis of variance). CONCLUSIONS Androgen ablation and alpha-blockade do not appear to alter the histologic characteristics of prostate stroma in men with symptomatic benign prostatic hyperplasia. However, contractile protein gene expression in stromal SM cells is significantly altered after alpha-blockade. These data suggest that, in addition to the simple relaxation of muscle tone, alpha-blocking agents may affect the phenotypic expression of contractile proteins in prostate SM cells.

SMOOTH MUSCLE MYOSIN HEAVY CHAINS ARE DEVELOPMENTALLY REGULATED IN THE RABBIT BLADDER

PURPOSE In smooth muscle (SM), myosin heavy chain (MHC) is expressed predominantly as two isoforms, SM1 and SM2, which are encoded by a single gene and expressed by alternative splicing mechanisms. Although functional differences of these isoforms are unknown, changes in SM1/SM2 ratio have been reported in various pathophysiologic conditions. We analyzed MHC composition of bladder detrusor SM from rabbits of different ages to determine whether SM1 and SM2 isoform expressions are developmentally regulated. MATERIALS AND METHODS Rabbit bladders on the -11, -4, 1, 7, 14, 21, and 90th days of life were analyzed for SM MHC isoform expression at protein and mRNA levels. Porous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), S1 protection assay, and histological analysis were employed. RESULTS The predominant MHC isoform in fetal and neonatal bladders was SM1. In the third postnatal week, the SM1/SM2 ratio decreased from 2.3 to 1.0. A stable SM1/SM2 ratio of 0.6 was observed in the adult animal. Although expression of SM1 mRNA was 2.6-fold greater than that of SM2 in the fetus, the relative amount of SM2 mRNA increased rapidly after birth and remained the predominant isoform throughout adult life. Developmental changes in relative amounts of SM1 and SM2 protein in bladder tissues were virtually identical to those of SM1 and SM2 mRNA. SM cell growth and disappearance of primitive mesenchyme from the bladder occurred concomitantly with the MHC isoform shift. CONCLUSIONS The parallel temporal course of MHC mRNA and protein isoform levels suggests detrusor SM MHC expression may be developmentally regulated at the mRNA level.

The use of chemokine-releasing tissue engineering scaffolds in a model of inflammatory response-mediated melanoma cancer metastasis

Inflammatory responses and associated products have been implicated in cancer metastasis. However, the relationship between these two processes is uncertain due to the lack of a suitable model. Taking advantage of localized and controllable inflammatory responses induced by biomaterial implantation and the capability of tissue scaffolds to release a wide variety of chemokines, we report a novel system for studying the molecular mechanisms of inflammation-mediated cancer metastasis. The animal model is comprised of an initial subcutaneous implantation of biomaterial microspheres which prompt localized inflammatory responses, followed by the transplantation of metastatic cancer cells into the peritoneal cavity or blood circulation. Histological results demonstrated that substantial numbers of B16F10 cells were recruited to the site nearby biomaterial implants. There was a strong correlation between the degree of biomaterial-mediated inflammatory responses and number of recruited cancer cells. Inflammation-mediated cancer cell migration was inhibited by small molecule inhibitors of CXCR4 but not by neutralizing antibody against CCL21. Using chemokine-releasing scaffolds, further studies were carried out to explore the possibility of enhancing cancer cell recruitment. Interestingly, erythropoietin (EPO) releasing scaffolds, but not stromal cell-derived factor-1α-releasing scaffolds, were found to accumulate substantially more melanoma cells than controls. Rather unexpectedly, perhaps by indirectly reducing circulating cancer cells, mice implanted with EPO-releasing scaffolds had ~30% longer life span than other groups. These results suggest that chemokine-releasing scaffolds may potentially function as implantable cancer traps and serve as powerful tools for studying cancer distraction and even selective annihilation of circulating metastatic cancer cells.

Molecular Aspects of Bladder Outlet Obstruction

In an animal model of obstruction, increasing load induces significant smooth muscle hypertrophy which is associated with a down-regulation of myosin heavy chain expression. This undoubtedly contributes to the decreased smooth muscle contractility seen in this model. Moreover, obstruction-induced hypertrophy leads to the development of a dedifferentiated smooth muscle phenotype, as evidenced by a revision of the cell to fetal (of non-muscle) gene expression patterns. Similar alterations are seen in atherosclerotic vessels and other pathologic smooth muscle systems. In these systems, dedifferentiation is also associated with significant alterations in extracellular matrix expression. It seems likely that obstruction in the bladder induces dedifferentiation of the smooth muscle cell which alters contractility as well as extracellular matrix expression, leading to altered bladder performance and decreased compliance.

A Comprehensive Approach Toward Novel Serum Biomarkers for Benign Prostatic Hyperplasia: The MPSA Consortium

PURPOSE Clinical benign prostatic hyperplasia is primarily diagnosed based on a diverse array of progressive lower urinary tract symptoms and is likely distinct from histological benign prostatic hyperplasia, which is detected by the presence of nonmalignant proliferation of prostate cells but may or may not be associated with symptoms. Pharmacological management of lower urinary tract symptoms has emerged as an effective initial treatment for clinical benign prostatic hyperplasia due to the introduction of new drug therapies shown to be effective in recent large clinical trials. Despite advances in symptom management and research into disease pathology, diagnostic strategies for the prediction of benign prostatic hyperplasia progression and response to drug modalities are lacking, and questions remain as to the molecular differences underlying clinical (symptomatic) vs histological (nonsymptomatic) benign prostatic hyperplasia. MATERIALS AND METHODS As part of the Medical Therapy of Prostatic Symptoms (MTOPS) clinical trial, which demonstrated the effectiveness of combination drug therapy in slowing benign prostatic hyperplasia progression, an archive of biological specimens linked to clinical data was collected for future profiling of disease pathology and changes associated with response to drug therapy. The MTOPS Prostatic Samples Analysis (MPSA) Consortium was established to identify and validate molecular markers that may better define benign prostatic hyperplasia related pathologies, identify risk of progression of lower urinary tract symptoms, and predict response to drug therapy using the MTOPS archive. The cooperating MPSA Biomarker Discovery Sites and Pathology Coordinating Center use diverse methodologies and scientific approaches as well as unique expertise to address the goals of the Consortium. RESULTS To date the MPSA has identified a number of promising biomarkers as well as other molecular and cellular changes associated with benign prostatic hyperplasia. CONCLUSIONS These findings and ongoing Consortium discovery efforts have the potential to provide a greater understanding of the defects underlying disease pathology, and may lead to the development of early and more effective pharmacological treatment strategies for benign prostatic hyperplasia.