Valproic Acid Prodrug Affects Selective Markers, Augments Doxorubicin Anticancer Activity and Attenuates Its Toxicity in a Murine Model of Aggressive Breast Cancer.

We studied the unique inhibitor of the histone deacetylases (HDAC) valproate-valpromide of acyclovir (AN446) that upon metabolic degradation release the HDAC inhibitor (HDACI) valproic acid (VPA). Among the HDAC inhibitors that we have tested, only AN446, and to a lesser extent VPA, synergized with doxorubicin (Dox) anti-cancer activity. Romidepsin (Rom) was additive and the other HDACIs tested were antagonistic. These findings led us to test and compare the anticancer activities of AN446, VPA, and Rom with and without Dox in the 4T1 triple-negative breast cancer murine model. A dose of 4 mg/kg once a week of Dox had no significant effect on tumor growth. Rom was toxic, and when added to Dox the toxicity intensified. AN446, AN446 + Dox, and VPA + Dox suppressed tumor growth. AN446 and AN446 + Dox were the best inhibitory treatments for tumor fibrosis, which promotes tumor growth and metastasis. Dox increased fibrosis in the heart and kidneys, disrupting their function. AN446 most effectively suppressed Dox-induced fibrosis in these organs and protected their function. AN446 and AN446 + Dox treatments were the most effective inhibitors of metastasis to the lungs, as measured by the gap area. Genes that control and regulate tumor growth, DNA damage and repair, reactive oxygen production, and generation of inflammation were examined as potential therapeutic targets. AN446 affected their expression in a tissue-dependent manner, resulting in augmenting the anticancer effect of Dox while reducing its toxicity. The specific therapeutic targets that emerged from this study are discussed.

The Transition from Gastric Intestinal Metaplasia to Gastric Cancer Involves POPDC1 and POPDC3 Downregulation.

Intestinal metaplasia (IM) is an intermediate step in the progression from premalignant to malignant stages of gastric cancer (GC). The Popeye domain containing (POPDC) gene family encodes three transmembrane proteins, POPDC1, POPDC2, and POPDC3, initially described in muscles and later in epithelial and other cells, where they function in cell-cell interaction, and cell migration. POPDC1 and POPDC3 downregulation was described in several tumors, including colon and gastric cancers. We questioned whether IM-to-GC transition involves POPDC gene dysregulation. Gastric endoscopic biopsies of normal, IM, and GC patients were examined for expression levels of POPDC1-3 and several suggested IM biomarkers, using immunohistochemistry and qPCR. Immunostaining indicated lower POPDC1 and POPDC3 labeling in IM compared with normal tissues. Significantly lower POPDC1 and POPDC3 mRNA levels were measured in IM and GC biopsies and in GC-derived cell lines. The reduction in focal IM was smaller than in extensive IM that resembled GC tissues. POPDC1 and POPDC3 transcript levels were highly correlated with each other and inversely correlated with LGR5, OLFM4, CDX2, and several mucin transcripts. The association of POPDC1 and POPDC3 downregulation with IM-to-GC transition implicates a role in tumor suppression and highlights them as potential biomarkers for GC progression and prospective treatment targets.

Farewell to Professor David Yaffe - A pillar of the myogenesis field.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel's War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a "David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe's earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

Cardioprotection by AN-7, a prodrug of the histone deacetylase inhibitor butyric acid: Selective activity in hypoxic cardiomyocytes and cardiofibroblasts.

The anticancer prodrug butyroyloxymethyl diethylphosphate (AN-7), upon metabolic hydrolysis, releases the histone deacetylase inhibitor butyric acid and imparts histone hyperacetylation. We have shown previously that AN-7 increases doxorubicin-induced cancer cell death and reduces doxorubicin toxicity and hypoxic damage to the heart and cardiomyocytes. The cardiofibroblasts remain unprotected against both insults. Herein we examined the selective effect of AN-7 on hypoxic cardiomyocytes and cardiofibroblasts and investigated mechanisms underlying the cell specific response. Hypoxic cardiomyocytes and cardiofibroblasts or H2O2-treated H9c2 cardiomyoblasts, were treated with AN-7 and cell damage and death were evaluated as well as cell signaling pathways and the expression levels of heme oxygenase-1 (HO-1). AN-7 diminished hypoxia-induced mitochondrial damage and cell death in hypoxic cardiomyocytes and reduced hydrogen peroxide damage in H9c2 cells while increasing cell injury and death in hypoxic cardiofibroblasts. In the cell line, AN-7 induced Akt and ERK survival pathway activation in a kinase-specific manner including phosphorylation of the respective downstream targets, GSK-3ß and BAD. Hypoxic cardiomyocytes responded to AN-7 treatment by enhanced phosphorylation of Akt, ERK, GSK-3ß and BAD and a significant 6-fold elevation in HO-1 levels. In hypoxic cardiofibroblasts, AN-7 did not activate Akt and ERK beyond the effect of hypoxia alone and induced a limited (~1.5-fold) increase in HO-1. The cell specific differences in kinase activation and in heme oxygenase-1 upregulation may explain, at least in part, the disparate outcome of AN-7 treatment in hypoxic cardiomyocytes and hypoxic cardiofibroblasts.

Extracellular-like matrices and leukaemia inhibitory factor for in vitro culture of human primordial follicles.

The possibility of maturing human primordial follicles in vitro would assist fertility restoration without the danger of reseeding malignancies. Leukaemia inhibitory factor (LIF) and certain culture matrices may promote human follicular growth. The present study compared human primordial follicular growth on novel culture matrices, namely human recombinant vitronectin (hrVit), small intestine submucosa (SIS), alginate scaffolds and human recombinant virgin collagen bioengineered in tobacco plant lines (CollPlant). The frozen-thawed ovarian samples that were used had been obtained from girls or young women undergoing fertility preservation. In the first part of the study, 20 samples were cultured for 6 days on hrVit or SIS with basic culture medium alone or supplemented with one of two concentrations of LIF (10ngmL-1 and 100ngmL-1), with and without LIF-neutralising antibody. In the second part of the study, 15 samples were cultured for 6 days on alginate scaffolds or CollPlant matrices with basic culture medium. Follicular development was assessed by follicular counts and classification, Ki67 immunohistochemistry and 17ß-oestradiol and anti-Müllerian hormone measurements in spent media samples. Primordial follicular growth was not enhanced by LIF. Despite some significant differences among the four matrices, none appeared to have a clear advantage, apart from significantly more Ki67-stained follicles on alginate and CollPlant matrices. Further studies of other culture matrices and medium supplements are needed to obtain an optimal system.

Short-term exposure of human ovarian follicles to cyclophosphamide metabolites seems to promote follicular activation in vitro.

How chemotherapy affects dormant ovarian primordial follicles is unclear. The 'burnout' theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using 4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24-48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17ß-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured (P = 0.04 to P < 0.0005) and uncultured controls (P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twicethe levels of AMH and 17ß-oestradiol in treated compared with untreated samples (AMH with 4hc 3 µmol/l; P = 0.04). All follicles stained positively for AMHincluded treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro. Study findings support the 'burnout' theory as the mechanism of chemotherapy-induced ovarian toxicity.

Popdc1/Bves Functions in the Preservation of Cardiomyocyte Viability While Affecting Rac1 Activity and Bnip3 Expression.

The Popeye domain containing1, also called Bves (Popdc1/Bves), is a transmembrane protein that functions in muscle regeneration, heart rate regulation, hypoxia tolerance, and ischemia preconditioning. The expression of Popdc1/Bves is elevated in cardiomyocytes maintained in serum free defined medium. We hypothesized that Popdc1/Bves is important for cardiomyocyte survival under the stress of serum deprivation and investigated the mechanisms involved. A deficit in Popdc1/Bves, achieved by siRNA-mediated gene silencing, results in cardiomyocyte injury and death, upregulation of the pro-apoptotic protein Bcl-2/adenovirus E1B 19-kDa interacting protein3 (Bnip3), as well as reduction in Rac1-GTPase activity and in Akt phosphorylation. Combined Popdc1/Bves and Bnip3 silencing attenuated cell injury and prevented Bnip3 upregulation induced by the silencing of Popdc1/Bves alone. Chromatin immunoprecipitation indicated an increased binding of the transcription factor FoxO3 to the Bnip3 promoter although augmentation of FoxO3 in the nuclei was not detected. By contrast, the transcription factor NFκB was excluded from the nuclei of Popdc1/Bves deficient cardiomyocytes and exhibited decreased binding to the Bnip3 promoter. The data indicates that Popdc1/Bves plays a role in the preservation of cardiomyocyte viability under serum deficiency through the alteration of Rac1 activity and the regulation of Bnip3 expression by FoxO3 and NFκB transcription factors pointing to Popdc1/Bves as a potential target to enhance heart protection. J. Cell. Biochem. 118: 1505-1517, 2017. © 2016 Wiley Periodicals, Inc.

Popeye domain containing 1 (Popdc1/Bves) is a caveolae-associated protein involved in ischemia tolerance.

Popeye domain containing1 (Popdc1), also named Bves, is an evolutionary conserved membrane protein. Despite its high expression level in the heart little is known about its membrane localization and cardiac functions. The study examined the hypothesis that Popdc1 might be associated with the caveolae and play a role in myocardial ischemia tolerance. To address these issues, we analyzed hearts and cardiomyocytes of wild type and Popdc1-null mice. Immunoconfocal microscopy revealed co-localization of Popdc1 with caveolin3 in the sarcolemma, intercalated discs and T-tubules and with costameric vinculin. Popdc1 was co-immunoprecipitated with caveolin3 from cardiomyocytes and from transfected COS7 cells and was co-sedimented with caveolin3 in equilibrium density gradients. Caveolae disruption by methyl-ß-cyclodextrin or by ischemia/reperfusion (I/R) abolished the cellular co-localization of Popdc1 with caveolin3 and modified their density co-sedimentation. The caveolin3-rich fractions of Popdc1-null hearts redistributed to fractions of lower buoyant density. Electron microscopy showed a statistically significant 70% reduction in caveolae number and a 12% increase in the average diameter of the remaining caveolae in the mutant hearts. In accordance with these changes, Popdc1-null cardiomyocytes displayed impaired [Ca(+2)]i transients, increased vulnerability to oxidative stress and no pharmacologic preconditioning. In addition, induction of I/R injury to Langendorff-perfused hearts indicated a significantly lower functional recovery in the mutant compared with wild type hearts while their infarct size was larger. No improvement in functional recovery was observed in Popdc1-null hearts following ischemic preconditioning. The results indicate that Popdc1 is a caveolae-associated protein important for the preservation of caveolae structural and functional integrity and for heart protection.

Fibroblast growth factor 10 in human ovaries.

The expression of fibroblast growth factor 10 (FGF-10) has not been studied in human ovarian cortical follicles. The aim of the present study was to investigate the expression of FGF-10 in preantral follicles from fetuses, girls and women. Ovarian samples were obtained from 14 human fetuses at 21-33 gestational weeks and from 35 girls and women aged 5-39 years. The specimens were prepared for detection of the FGF-10 protein by immunohistochemistry. Reverse-transcription PCR was applied to ovarian extracts to identify FGF-10 mRNA transcripts. In fetal tissue, the FGF-10 protein was detected in oocytes in 50% of the samples and in granulosa cells in 30%. In ovarian tissue from girls and women, the FGF-10 protein was detected in oocytes and granulosa cells in all samples. FGF-10 mRNA transcripts were present in all adult and fetal samples tested. The identification of FGF-10 at both the protein and mRNA levels suggests that FGF-10 may contribute to human preantral follicle development.

Vasoactive intestinal peptide and its receptors in human ovarian cortical follicles.

BACKGROUND: Ovarian cryopreservation is one option for fertility preservation in patients with cancer. The danger of reseeding malignancies could be eliminated by in vitro maturation of primordial follicles from the frozen-thawed tissue. However, the development of this system is hindered by uncertainties regarding factors that activate primordial follicles. Neuronal growth factors such as vasoactive intestinal peptide (VIP) play important roles in early mammalian folliculogenesis. There are no data on the expression of VIP and its vasoactive intestinal peptide pituitary adenylate cyclase 1 and 2 receptors (VPAC1-R and VPAC2-R) in human preantral follicles. METHODOLOGY/PRINCIPAL FINDINGS: Tissue samples from 14 human fetal ovaries and 40 ovaries from girls/women were prepared to test for the expression of VIP, VPAC1-R, and VPAC2-R on the protein (immunohistochemisty) and mRNA (reverse transcription polymerase chain reaction) levels. Immunohistochemistry staining was mostly weak, especially in fetal samples. The VIP protein was identified in oocytes and granulosa cells (GCs) in the fetal samples from 22 gestational weeks (GW) onwards. In girls/women, VIP follicular staining (oocytes and GCs) was identified in 45% of samples. VPAC1-R protein was identified in follicles in all fetal samples from 22GW onwards and in 63% of the samples from girls/women (GC staining only in 40%). VPAC2-R protein was identified in follicles in 33% of fetal samples and 47% of the samples from girls/women. The mRNA transcripts for VIP, VPAC1-R, and VPAC2-R were identified in ovarian extracts from fetuses and women. CONCLUSIONS: VIP and its two receptors are expressed in human ovarian preantral follicles. However, their weak staining suggests they have limited roles in early follicular growth. To elucidate if VIP activates human primordial follicles, it should be added to the culture medium.

Disparate impact of butyroyloxymethyl diethylphosphate (AN-7), a histone deacetylase inhibitor, and doxorubicin in mice bearing a mammary tumor.

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.

The histone deacetylase inhibitor butyroyloxymethyl diethylphosphate (AN-7) protects normal cells against toxicity of anticancer agents while augmenting their anticancer activity.

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) has been shown to synergize doxorubicin (Dox) anticancer activity while attenuating its cardiotoxicity. In this study we further explored the selectivity of AN-7's action in several cancer and normal cells treated with anticancer agents. The cells studied were murine mammary 4T1, human breast T47D and glioblastoma U251 cancer cell lines, neonatal rat cardiomyocytes, cardiofibroblasts and astrocytes, and immortalized cardiomyocyte H9C2 cells. Cell death, ROS production and changes in protein expression were measured and in vivo effects were evaluated in Balb-c mice. AN-7 synergized Dox and anti-HER2 cytotoxicity against mammary carcinoma cells with combination indices of 0.74 and 0.79, respectively, while it protected cardiomyocytes against their toxicity. Additionally AN-7 protected astrocytes from Dox-cytoxicity. Cell-type specific changes in the expression of proteins controlling survival, angiogenesis and inflammation by AN-7 or AN-7+Dox were observed. In mice, the protective effect of AN-7 against Dox cardiotoxicity was associated with a reduction in inflammatory factors. In summary, AN-7 augmented the anticancer activity of Dox and anti-HER2 and attenuated their toxicity against normal cells. AN-7 modulation of c-Myc, thrombospondin-1, lo-FGF-2 and other proteins were cell type specific. The effects of AN-7, Dox and their combination were preserved in vivo indicating the potential benefit of combining AN-7 and Dox for clinical use.

Popeye domain-containing 1 is down-regulated in failing human hearts.

Congestive heart failure, a complex disease of heterogeneous etiology, involves alterations in the expression of multiple genes. The Popeye domain-containing (POPDC) family of three novel muscle-restricted genes (POPDC1-3) is evolutionarily conserved and developmentally regulated. In mice, POPDC1 has been shown to play an important role in skeletal and cardiac muscles subjected to injury or stress. However, it has never been explored in human hearts. In biopsies from non-failing and failing human hearts, we examined the cellular distribution of POPDC1 as well as the expression patterns of POPDC1-3 mRNAs. POPDC1 was visualized by immunohistochemistry and estimated by Western immunoblotting. The mRNA levels of POPDC1-3 and ß myosin heavy chain (MYHC7) were assessed using reverse transcription/quantitative polymerase chain reaction. POPDC1 was predominantly localized in the sarcolemma with an enhanced expression in the intercalated discs. In failing hearts, many cardiomyocytes appeared deformed and POPDC1 labeling was deranged. The three POPDC mRNAs were expressed in the four heart chambers with higher transcript levels in the ventricles compared to the atria. Heart failure concurred with reduced levels of POPDC1 mRNA and protein in the left ventricle. Correlation analyses of mRNA levels among the failing heart specimens indicated the coordinated regulation of POPDC1 with POPDC3 and of POPDC2 with MYHC7. It can be concluded that POPDC gene expression is modified in end-stage heart failure in humans in a manner suggesting regulatory and/or functional differences between the three family members and that POPDC1 is particularly susceptible to this condition.

Exercise training alters the molecular response to myocardial infarction.

PURPOSE: We and others have shown that swimming exercise training performed before irreversible coronary occlusion improves the outcome of the heart injury and alters gene expression at the remodeling phase. The purpose of the current study was to identify temporal changes in the molecular response to myocardial infraction of prior exercise trained rats during the acute, the subacute, and the chronic phases postinfarction. METHODS: Rats underwent a 7-wk swimming or sedentary protocol and were subjected to surgical induction of acute myocardial infarction (MI). Hearts were removed before and at 4 h, 2 d, and 4 wk after surgery. RNA extracted from the surviving myocardium of the MI hearts or from corresponding tissues in the non-MI hearts was subjected to multitranscript profiling. Results for representative transcripts were validated by reverse transcription and quantitative polymerase chain reaction amplification. RESULTS: Global analysis of the 3686 detected transcripts generated a two-branch dendrogram that distinguished the pre-MI and the 4-h groups from the 2-d and the 4-wk groups and indicated that early after MI, the impact of infarction on the genes expressed overrides the training effect, whereas at 4 wk, the exercised hearts differ markedly from the nonexercised. Clustering the 1500 genes that showed the highest variance over time indicated differential expression of transcription regulators and proapoptotic genes 4 h and 2 d after MI and of stress-related and profibrotic genes 4 wk later in the exercised compared with sedentary hearts. CONCLUSION: Swimming exercise training conducted before acute MI reprograms the surviving myocardium for altered molecular response to MI that explains, in part, the protected cardiac phenotype of the exercised animals.

The Popdc gene family in the rat: molecular cloning, characterization and expression analysis in the heart and cultured cardiomyocytes.

Three Popeye domain-containing (Popdc 1-3) family-members are known in vertebrates. Their exact function is as yet unknown although involvement in cell adhesion has been suggested. We report herein sequencing of the rat Popdc 1-3 cDNAs that show high homology to other vertebrate orthologs and are expressed primarily in the heart and skeletal muscles. Popdc2 splice variants were identified, with Popdc2C showing a distinctive age-dependent decline. In isolated cardiomyocytes, Popdc genes were negatively regulated by serum, an effect that was reversed by EGFR-kinase inhibition, suggesting an EGFR-dependent modulation of Popdc gene expression.

Expression of procollagen C-proteinase enhancer-1 in the remodeling rat heart is stimulated by aldosterone.

Excessive collagen deposition is a common complication of myocardial infarction that causes progressive heart disease. Several pro-fibrotic cytokines and hormones, including aldosterone, control this process. Procollagen processing by procollagen C-proteinase(s) is critical for collagen deposition and is potentiated by procollagen C-proteinase enhancer proteins (PCPEs). We have shown previously that, in addition to stimulation of collagen I expression, aldosterone increases PCPE-1 expression in cultured heart fibroblasts. The present study was designed to examine whether aldosterone acts similarly in vivo. Rats underwent coronary artery ligation to induce myocardial infarction. They were then left either untreated (control) or treated with spironolactone (an aldosterone receptor antagonist) for 5 weeks when they were sacrificed and their hearts removed for analysis. In situ hybridization co-localized PCPE-1 and collagen I mRNAs to fibroblasts surrounding the scar region and adjacent blood vessels. The levels of both transcripts in the remodeling myocardium of untreated rats increased twofold as compared to sham-operated controls, an increase greatly reduced by spironolactone. Correspondingly, a 2-5 fold increase in PCPE-1 and collagen I was observed in the hearts of untreated rats as compared to both the spironolactone-treated and sham-operated controls. The results establish aldosterone as a physiological stimulator of PCPE-1 expression in the remodeling myocardium after infarction. Since PCPE-1 itself is a positive regulator of collagen deposition, this finding suggests PCPE-1 as a new potential target for intervention with cardiac fibrosis.

Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses.

OBJECTIVE: To investigate the immunocytochemical expression and presence of mRNA transcripts for basic fibroblast growth factor (bFGF) and its four receptors (FGFR-1, -2, -3, and -4) in ovaries from human adults and fetuses. DESIGN: Immunocytochemical and reverse transcription polymerase chain reaction (RT-PCR) study. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Nine women and girls undergoing laparoscopic ovarian biopsy and 26 women undergoing second- and third-trimester pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microscopic morphometric analysis, immunocytochemistry for bFGF and its receptors, and RT-PCR analyses. RESULT(S): The proteins for bFGF, FGFR-2, FGFR-3, and FGFR-4 were identified in oocytes of all follicular classes. Immunocytochemical expression of bFGF and its receptors was detected in granulosa cells of follicles from adolescents/women but not from fetuses. There was no immunocytochemical expression of FGFR-1. Transcripts of bFGF and its four receptors were identified by RT-PCR in all samples. CONCLUSION(S): The expression of bFGF and its receptors in human ovaries suggests that bFGF might have a role in early folliculogenesis.

Prior exercise training improves the outcome of acute myocardial infarction in the rat. Heart structure, function, and gene expression.

OBJECTIVES: The aim of this research was to investigate the structural, functional, and molecular features of the remodeling heart in prior swim-trained infarcted rats. BACKGROUND: Physical exercise training is a known protective factor against cardiovascular morbidity and mortality. The structural and molecular aspects underlying this protection in the remodeling heart have not been investigated. METHODS: After seven weeks of swimming exercise training, rats underwent surgical ligation of the left coronary artery followed by a four-week sedentary period. Untrained control rats underwent the same surgical protocol. Left ventricular function was assessed by echocardiography four weeks after infarction, and hearts were sampled for histological and molecular analysis. Ribonucleic acid from the surviving left ventricle was analyzed by complementary deoxyribonucleic acid arrays followed by Northern blotting or quantitative reverse transcription polymerase chain reaction of selected messenger ribonucleic acids (mRNAs). RESULTS: Scar area was 1.6-fold smaller (p = 0.0002), arteriolar density was 1.7-fold higher (p = 0.0002), and left ventricular shortening fraction was 1.9-fold higher (p = 0.003) in the exercise-trained compared with sedentary hearts. Eleven genes whose expression level varied by at least +/-1.5-fold distinguished the prior exercised rats from their sedentary counterparts. Compared with sedentary, the exercised hearts displayed 9- and 2.4-times lower levels of atrial natriuretic peptide and aldolase mRNA (p = 0.03 and 0.04, respectively), and a 2.7- and 1.9-fold higher abundance of cytochrome c-oxidase and fatty acid binding protein, respectively (p < 0.03, each). CONCLUSIONS: Swimming exercise training before acute myocardial infarction reduces scar size, increases arteriole density, and manifests adaptation of stress- and energy-metabolism-related genes that may contribute to the improved heart function observed during remodeling.

Expression of stem cell factor and its receptor in human fetal and adult ovaries.

OBJECTIVE: To investigate the immunocytochemical expression and presence of mRNA transcripts of stem cell factor (SCF) and its receptor (SCF-R) in ovaries from human adults and fetuses. DESIGN: Immunocytochemical and reverse transcription polymerase chain reaction (RT-PCR) study. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Seven women and girls undergoing laparoscopic ovarian biopsy and 13 women undergoing second and third trimester pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microscopic morphometric analysis, immunocytochemistry for SCF and SCF-R, and RT-PCR analyses. RESULT(S): There was strong to moderate immunocytochemical staining for SCF and its receptor in oocytes from primordial stages onward, but not in granulosa cells, in both fetal and adult ovarian samples. Transcripts of SCF and SCF-R RNA were detected by RT-PCR analyses for SCF and SCF-R. CONCLUSION(S): The expression of SCF and its receptor in ovarian tissue from fetuses and women suggests a possible role of SCF in growth initiation of human primordial follicles.

Expression of procollagen C-proteinase enhancer in cultured rat heart fibroblasts: evidence for co-regulation with type I collagen.

Procollagen processing by procollagen C-proteinase (PCP) is an important step in collagen deposition. This reaction is stimulated by another glycoprotein, known as PCP enhancer. The objective of this study was to identify factors that regulate the expression of PCP enhancer in cardiac fibroblasts and examine possible correlation with collagen expression. Rat heart fibroblasts were cultured in the presence or absence of three known stimulators of collagen synthesis: ascorbic acid, TGF-beta, and aldosterone. The mRNA and protein levels of PCP enhancer and collagen type I were each assessed using Northern and Western blotting, respectively. Expression of PCP was assessed by RT-PCR and its activity in the culture media was determined using radioactive procollagen as the substrate. The levels of PCP enhancer mRNA increased 1.5- to 2-fold in response to ascorbate, TGF-beta, or aldosterone. This increase was paralleled by an up to fourfold increase in the level of the pro alpha1(I) collagen chain transcript and was accompanied by a marked increase in the levels of the respective proteins in the culture media. PCP activity in the culture media was also increased, apparently, without effect on its expression. These results indicate that expression of PCP enhancer in cultured rat heart fibroblasts is coordinated with that of collagen. The observed augmentation of PCP activity may be a consequence of the increase in the levels of PCP enhancer in the culture media.