Perturbations of genes essential for Müllerian duct and Wölffian duct development in Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

LncRNA RP11-301G19.1 is required for the maintenance of vascular smooth muscle cell contractile phenotype via sponging miR-17-5P/ATOH8 axis.

Long noncoding RNAs (LncRNAs) play essential roles in regulating gene expression in various biological processes. However, the function of lncRNAs in vascular smooth muscle cell (VSMC) transformation remains to be explained. In this work, we discover that a new bone marrow protein (BMP) signaling target, lncRNA RP11-301G19.1, is significantly induced in BMP7-treated VSMCs through lncRNA microarray analysis. Addition of BMP signaling inhibitor LDN-193189 attenuates the expression of ACTA2 and SM-22α, as well as the mRNA level of RP11-301G19.1. Furthermore, lncRNA RP11-301G19.1 is critical to the VSMC differentiation and is directly activated by SMAD1/9. Mechanistically, knocking down of RP11-301G19.1 leads to the decrease of ATOH8, another BMP target, while the forced expression of RP11-301G19.1 reactivates ATOH8. In addition, miR-17-5p, a miRNA negatively regulated by BMP-7, contains predicted binding sites for lncRNA RP11-301G19.1 and ATOH8 3'UTR. Accordingly, overexpression of miR-17-5p decreases the levels of them. Together, our results revealed the role of lncRNA RP11-301G19.1 as a miRNA sponge to upregulate ATOH8 in VSMC phenotype transformation.

A systematic review and cross-database analysis of single nucleotide polymorphisms underlying hip morphology and osteoarthritis reveals shared mechanisms.

OBJECTIVE: Understanding the mechanisms of hip disease, such as osteoarthritis (OA), is crucial to advance their treatment. Such hip diseases often involve specific morphological changes. Genetic variations, called single nucleotide polymorphisms (SNPs), influence various hip morphological parameters. This study investigated the biological relevance of SNPs correlated to hip morphology in genome-wide association studies (GWAS). The SNP-associated genes were compared to genes associated with OA in other joints, aiming to see if the same genes play a role in both hip development and the risk of OA in other lower limb joints. METHODOLOGY: A systematic literature review was conducted to identify SNPs correlated with hip morphology, based on the Population, Intervention, Comparison, Outcome, and Study (PICOS) framework. Afterwards, Gene Ontology (GO) analysis was performed, using EnrichR, on the SNP-associated genes and compared with non-hip OA-associated genes, across different databases. RESULTS: Reviewing 49 GWAS identified 436 SNPs associated with hip joint morphology, encompassing variance in bone size, structure and shape. Among the SNP-associated genes, SOX9 plays a pivotal role in size, GDF5 impacts bone structure, and BMP7 affects shape. Overall, skeletal system development, regulation of cell differentiation, and chondrocyte differentiation emerged as crucial processes influencing hip morphology. Eighteen percent of GWAS-identified genes related to hip morphology were also associated with non-hip OA. CONCLUSION: Our findings indicate the existence of multiple shared genetic mechanisms across hip morphology and OA, highlighting the necessity for more extensive research in this area, as in contrast to the hip, the genetic background on knee or foot morphology remains largely understudied.

BMP heterodimers signal via distinct type I receptor class functions.

Heterodimeric TGF-ß ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex.

Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/ß-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast-adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/ß-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

Lead Decreases Bone Morphogenetic Protein-7 (BMP-7) Expression and Increases Renal Cell Carcinoma Growth in a Sex-Divergent Manner.

Both tissue and blood lead levels are elevated in renal cell carcinoma (RCC) patients. These studies assessed the impact of the subchronic lead challenge on the progression of RCC in vitro and in vivo. Lead challenge of Renca cells with 0.5 µM lead acetate for 10 consecutive passages decreased E-cadherin expression and cell aggregation. Proliferation, colony formation, and wound healing were increased. When lead-challenged cells were injected into mice, tumor size at day 21 was increased; interestingly, this increase was seen in male but not female mice. When mice were challenged with 32 ppm lead in drinking water for 20 weeks prior to tumor cell injection, there was an increase in tumor size in male, but not female, mice at day 21. To investigate the mechanism underlying the sex differences, the expression of sex hormone receptors in Renca cells was examined. Control Renca cells expressed estrogen receptor (ER) alpha but not ER beta or androgen receptor (AR), as assessed by qPCR, and the expression of ERα was increased in tumors in both sexes. In tumor samples harvested from lead-challenged cells, both ERα and AR were detected by qPCR, yet there was a significant decrease in AR seen in lead-challenged tumor cells from male mice only. This was paralleled by a plate-based array demonstrating the same sex difference in BMP-7 gene expression, which was also significantly decreased in tumors harvested from male but not female mice; this finding was validated by immunohistochemistry. A similar expression pattern was seen in tumors harvested from the mice challenged with lead in the drinking water. These data suggest that lead promotes RCC progression in a sex-dependent via a mechanism that may involve sex-divergent changes in BMP-7 expression.

High BMP7 expression is associated with poor prognosis in ovarian cancer.

Bone Morphogenetic Protein 7 (BMP7) is an extracellular signalling protein that belongs to the transforming growth factor-ß (TGF- ß) superfamily. Previous transcriptomic data suggested that BMP7 expression may be disrupted in ovarian carcinoma and may play an important role in the aggressiveness of the disease. However, the protein expression in patient tumours has not been well studied. The current study aimed to assess BMP7 protein expression in a large cohort of ovarian carcinoma patient tumour samples to establish its associations with different clinical endpoints. Ovarian carcinoma tissue samples from 575 patients who underwent surgery for different subtypes of ovarian cancer were used. BMP7 protein expression was analysed by immunohistochemistry using tissue microarray and full face tumour sections. High BMP7 expression is associated with aggressive ovarian cancer clinicopathological variables including advanced FIGO stage, high grade, residual disease and poor overall survival. Elevated cytoplasmic and nuclear BMP7 expression was significantly associated with advanced FIGO stage, high tumour grade, presence of residual tumours and high-grade serous carcinomas (p = 0.001, 0.005, 0.004, <0.001 and p < 0.001, <0.001, 0.002, 0.001 respectively). Increased cytoplasmic and nuclear BMP7 expression was also significantly associated with an adverse overall survival (p = 0.001 and 0.046 respectively). The study highlights the potential of BMP7 as a prognostic tool and as a potential novel target for ovarian cancer therapies to limit disease progression.

Protective effect of bone morphogenetic protein-7 induced differentiation of bone marrow mesenchymal stem cells in rat with acute spinal cord injury.

The principal aim of present study was to assess the therapeutic efficacy of bone morphogenetic protein-7 (BMP-7) induced differentiation of bone marrow mesenchymal stem cells (BMSCs) in a rat acute spinal cord injury (SCI) model. BMSCs were isolated from rats, and then divided into a control and a BMP-7 induction groups. The proliferation ability of BMSCs and glial cell markers were determined. Forty Sprague-Dawley (SD) rats were randomly divided into sham, SCI, BMSC, and BMP7 + BMSC groups (n = 10). Among these rats, the recovery of hind limb motor function, the pathological related markers, and motor evoked potentials (MEP) were identified. BMSCs differentiated into neuron-like cells after the introduction of exogenous BMP-7. Interestingly, the expression levels of MAP-2 and Nestin increased, whereas the expression level of GFAP decreased after the treatment with exogenous BMP-7. Furthermore, the Basso, Beattie, and Bresnahan (BBB) score reached 19.33 ± 0.58 in the BMP-7 + BMSC group at day 42. Nissl bodies in the model group were reduced compared to the sham group. After 42 days, in both the BMSC and BMP-7 + BMSC groups, the number of Nissl bodies increased. This is especially so for the number of Nissl bodies in the BMP-7 + BMSC group, which was more than that in the BMSC group. The expression of Tuj-1 and MBP in BMP-7 + BMSC group increased, whereas the expression of GFAP decreased. Moreover, the MEP waveform decreased significantly after surgery. Furthermore, the waveform was wider and the amplitude was higher in BMP-7 + BMSC group than that in BMSC group. BMP-7 promotes BMSC proliferation, induces the differentiation of BMSCsinto neuron-like cells, and inhibits the formation of glial scar. BMP-7 plays a confident role in the recovery of SCI rats.

Assessment of BMP7, SMAD4, and CDH1 Expression Profile and Regulatory miRNA-542-3p in Eutopic and Ectopic Endometrium of Women with Endometriosis.

Alterations in the expression of numerous genes and the miRNAs that are recognized as their regulators in the endometrial cells of women with endometriosis may disrupt the intracellular signaling pathways associated with epithelial-mesenchymal transition (EMT). So far, the functional role of BMP7 in endometrial physiology has been confirmed, especially in the context of fertility, but the role of the activation of a specific mechanism operating through the BMP-SMAD-CDH1 axis in the formation of endometrial lesions remains unexplored. The aim of this study was to evaluate the expression profile of miR-542-3p and the EMT markers (BMP7, SMAD4, CDH1) in matched eutopic endometrium (EUE) and ectopic endometrium (ECE) samples from women with endometriosis in relation to healthy women. The levels of expression of the studied genes and miRNA in peripheral blood mononuclear cells (PBMCs) obtained from women diagnosed with endometriosis and those without the disease were also evaluated. Fifty-four patients (n = 54: with endometriosis-n = 29 and without endometriosis-n = 25) were included in the study. A comparative analysis of the relative mean expression values (RQ) of the studied mRNA and miRNA assessed by RT-qPCR demonstrated downregulation of BMP7, SMAD4, and CDH1 expression in ectopic lesions and upregulation in the eutopic endometrium compared with the control group. In the eutopic tissue of women with endometriosis, miR-542-3p expression was similar to that of the control but significantly lower than in endometrial lesions. We also confirmed a trend towards a negative correlation between miR-542-3p and BMP7 in ectopic tissue, and in PBMC, a significant negative correlation of miR-542-3p with further BMP signaling genes, i.e., SMAD4 and CDH1, was observed. These results indicate that the miRNA selected by us may be a potential negative regulator of BMP7-SMAD4-CDH1 signaling associated with EMT. The different patterns of BMP7, SMAD4, and CDH1 gene expression in ECE, EUE, and the control endometrium observed by us suggests the loss of the endometrial epithelium phenotype in women with endometriosis and demonstrates their involvement in the pathogenesis and pathomechanism of this disease.

Effect of crocin and losartan on biochemical parameters and genes expression of FRMD3 and BMP7 in diabetic rats.

BACKGROUND: Diabetes is a multifactorial and growing disease, one of the severe complications of which is diabetic nephropathy (DN), which is the most common cause of chronic renal failure. FERM domain containing 3 (FRMD3) is responsible for maintaining the shape and integrity of nephron cells, and bone morphogenetic protein 7 (BMP7) helps maintain function and reduce kidney damage. This study aimed to evaluate the effect of crocin and losartan on biochemical parameters and the expression of FRMD3 and BMP7 genes in streptozotocin (STZ)-induced diabetic rats. METHODS: Forty male Wistar rats were randomly divided into five experimental groups as healthy, diabetic control (D), crocin, losartan, and diabetic rats treated with losartan-crocin (n = 8). A single dose of STZ (50 mg/kg intraperitoneally injection) was used to induce diabetes. Four weeks after induction of diabetes, rats received crocin (50 mg/kg) and losartan (25 mg/kg) daily for four weeks orally. Rats were sacrificed at the end of the intervention, and blood samples were taken to determine serum levels of glucose, urea, creatinine (Cr), malondialdehyde (MDA), and thiol. Real-time polymerase chain reaction (PCR) was used to assess the expression of the FRMD3 and BMP7 genes in the kidney samples. RESULTS: Diabetes induction increased serum levels of glucose, Cr, urea, MDA, and thiol, but decreased BMP7 and FRMD3 genes expression. Treatment with crocin and losartan decreased these biochemical parameters and increased the expression of the BMP7 and FRMD3 genes. DISCUSSION: Crocin may be a promising therapeutic agent for preventing and improving diabetes-related kidney disease due to its antidiabetic and antioxidant properties.

HOXA13 promotes liver regeneration through regulation of BMP-7.

In-depth knowledge of liver regeneration could facilitate the development of therapies for liver injury and liver failure. As a member of the homeobox superfamily, HOXA13 plays an important role in regulating tumorigenesis and development. However, the exact role of HOXA13 in liver regeneration remains unclear. In this study, we confirmed that HOXA13 promotes hepatocyte proliferation both in vivo and in vitro. HOXA13 was upregulated during liver regeneration, and its overexpression further accelerated hepatocyte proliferation and liver function recovery during liver regeneration. Furthermore, we found that HOXA13 promoted hepatocyte proliferation and liver regeneration by upregulating bone morphogenetic protein-7 (BMP-7) mRNA. These findings provide a new potential target for the treatment of liver failure.

Sperm-oviduct interaction: Differential gene expression of growth factors induced by sperm DNA fragmentation.

The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.

Activating Wnt/ß-Catenin Signaling in Osteocytes Promotes Osteogenic Differentiation of BMSCs through BMP-7.

Bone formation is critically needed in orthopedic clinical practice. We found that, bone morphogenetic protein-7 (BMP-7) gene expression was significantly increased in fractured mice, which activates canonical Wnt signaling exclusively in osteocytes. Wnt and BMP signaling appear to exhibit synergistic or antagonistic effects in different kinds of cells. However, the communication between Wnt/ß-catenin signaling and BMP signaling in osteocytes is almost unknown. Our study verified in vitro that BMP-7 expression was significantly increased when Wnt signaling was activated in osteocytes. Next, BMP-7 in osteocytes was overexpressed using an adenovirus, the osteogenesis of bone marrow stem cells (BMSCs) was enhanced, when cocultured with osteocytes. On the contrary, BMP-7 in osteocytes was silenced using an adenovirus, the osteogenesis of bone marrow stem cells (BMSCs) was weakened. In addition, the osteogenesis of BMSCs was no longer promoted by Wnt-activated osteocytes when BMP-7 was silenced. Therefore, the results showed that BMP-7 mediated the anabolic actions of Wnt/ß-catenin signaling in osteocytes. Our study provides new evidence for the clinical application of BMP-7-overexpressed osteocytes.

Neonatal nephron loss during active nephrogenesis results in altered expression of renal developmental genes and markers of kidney injury.

Preterm neonates are at a high risk for nephron loss under adverse clinical conditions. Renal damage potentially collides with postnatal nephrogenesis. Recent animal studies suggest that nephron loss within this vulnerable phase leads to renal damage later in life. Nephrogenic pathways are commonly reactivated after kidney injury supporting renal regeneration. We hypothesized that nephron loss during nephrogenesis affects renal development, which, in turn, impairs tissue repair after secondary injury. Neonates prior to 36 wk of gestation show an active nephrogenesis. In rats, nephrogenesis is ongoing until day 10 after birth. Mimicking the situation of severe nephron loss during nephrogenesis, male pups were uninephrectomized at day 1 of life (UNXd1). A second group of males was uninephrectomized at postnatal day 14 (UNXd14), after terminated nephrogenesis. Age-matched controls were sham operated. Three days after uninephrectomy transcriptional changes in the right kidney were analyzed by RNA-sequencing, followed by functional pathway analysis. In UNXd1, 1,182 genes were differentially regulated, but only 143 genes showed a regulation both in UNXd1 and UNXd14. The functional groups "renal development" and "kidney injury" were among the most differentially regulated groups and revealed distinctive alterations. Reduced expression of candidate genes concerning renal development (Bmp7, Gdnf, Pdgf-B, Wt1) and injury (nephrin, podocin, Tgf-ß1) were detected. The downregulation of Bmp7 and Gdnf persisted until day 28. In UNXd14, Six2 was upregulated and Pax2 was downregulated. We conclude that nephron loss during nephrogenesis affects renal development and induces a specific regulation of genes that might hinder tissue repair after secondary kidney injury.

BMP7 overexpression in adipose tissue induces white adipogenesis and improves insulin sensitivity in ob/ob mice.

BACKGROUND/OBJECTIVES: During obesity, hypertrophic enlargement of white adipose tissue (WAT) promotes ectopic lipid deposition and development of insulin resistance. In contrast, WAT hyperplasia is associated with preservation of insulin sensitivity. The complex network of factors that regulates white adipogenesis is not fully understood. Bone morphogenic protein 7 (BMP7) can induce brown adipogenesis, but its role on white adipogenesis remains to be elucidated. Here, we assessed BMP7-mediated effects on white adipogenesis in ob/ob mice. METHODS: BMP7 was overexpressed in either WAT or liver of ob/ob mice using adeno-associated viral (AAV) vectors. Analysis of gene expression, histological and morphometric alterations, and metabolites and hormones concentrations were carried out. RESULTS: Overexpression of BMP7 in adipocytes of subcutaneous and visceral WAT increased fat mass, the proportion of small-size adipocytes and the expression of adipogenic and mature adipocyte genes, suggesting induction of adipogenesis irrespective of fat depot. These changes were associated with reduced hepatic steatosis and improved insulin sensitivity. In contrast, liver-specific overproduction of BMP7 did not promote WAT hyperplasia despite BMP7 circulating levels were similar to those achieved after genetic engineering of WAT. CONCLUSIONS: This study unravels a new autocrine/paracrine role of BMP7 on white adipogenesis and highlights that BMP7 may modulate WAT plasticity and increase insulin sensitivity.

Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

Romidepsin hepatocellular carcinoma suppression in mice is associated with deregulated gene expression of bone morphogenetic protein and Notch signaling pathway components.

Recently, our group showed that Romidepsin, a histone deacetylase inhibitor (HDACi), suppressed diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in mice. In the present study, we investigated the effect of Romidepsin-treatment on gene expression levels of components of Bmp and Notch signaling pathways, which are both known to be aberrantly regulated in hepatocarcinogenesis. Total RNA from liver tissue samples and paraffin-embedded livers were retrieved from a recent experiment where C57BL/6 mice were treated with Romidepsin 10 months after DEN challenge and sacrificed 2 months later. RT qPCR was used for quantification of gene expression and immunohistochemistry for in situ protein detection. Regarding Bmp pathway, Romidepsin HCC-suppression was found to correlate significantly with Bmp2 and Bmp7 ligand up- and down-regulation, respectively. Intracellularly, Romidepsin-treated HCC mice exhibited a significant elevation of Bmp-inhibitor Smurf2 and Bmp-target gene Id3, as compared to the HCC untreated controls. Concerning Notch signaling, higher expression levels of ligands Jag1/Dll4, accompanied by a decreased expression of receptor Notch2, were identified in the Romidepsin-treated group. Τhe anti-oncogenic effect of Romidepsin, also correlated significantly with an increased expression of Hes1 target, as well as an up- and down-regulation of Klf4 and Sox9 transcription factors, respectively. Moreover, the cancer-related genes Snai2 and p21, known to be involved in many signaling pathways, including Bmp and Notch, were also found to be downregulated in Romidepsin-treated mice. Romidepsin HCC suppression is associated with gene expression deregulation of selective components of both Bmp and Notch signaling cascades.

Gremlin-1 is a key regulator of endothelial-to-mesenchymal transition in human pulmonary artery endothelial cells.

BACKGROUND: Endothelial-to-mesenchymal transition (EndMT) has been implicated in initiation and progression of pulmonary arterial hypertension (PAH). Gremlin-1 promotes vascular remodeling of PAH and mediates epithelial-mesenchymal transition, which is similar to EndMT. In the present study we investigated the potential role of gremlin-1 plays in EndMT of pulmonary artery endothelial cells (PAECs). METHODS: Immunofluorescence staining was performed to detect the expression of alpha smooth muscle actin (α-SMA) and von Willebrand factor (VWF). Migration and angiogenic responses of PAECs were determined by transwell assay and tube formation assay, respectively. Protein expression levels were determined by western blotting. RESULTS: Gremlin-1 induced EndMT of PAECs in a phospho-smad2/3-dependent manner. This was characterized by the loss of platelet endothelial cell adhesion molecule 1 and an increase in protein levels of a-SMA, nerve-cadherin, and matrix metalloproteinase 2. It was also determined that gremlin-1 facilitated the migration and angiogenic responses of PAECs in a dose-dependent manner. Bone morphogenetic protein 7 (BMP-7) was found to attenuate gremlin-1-mediated EndMT, migration and angiogenesis of PAECs by inducing phosphorylation of Smad1/5/8 and suppressing phosphorylation of Smad2/3. CONCLUSION: Gremlin-1 mediates EndMT in PAECs, and BMP-7 reverses gremlin-1-induced EndMT by an induction of p-Smad1/5/8 and suppression of p-Smad2/3.

Ultrasound triggered topical delivery of Bmp7 mRNA for white fat browning induction via engineered smart exosomes.

BACKGROUND: Efficient and topical delivery of drugs is essential for maximized efficacy and minimized toxicity. In this study, we aimed to design an exosome-based drug delivery platform endowed with the ability of escaping from phagocytosis at non-target organs and controllably releasing drugs at targeted location. RESULTS: The swtichable stealth coat CP05-TK-mPEG was synthesized and anchored onto exosomes through the interaction between peptide CP05 and exosomal surface marker CD63. Chlorin e6 (Ce6) was loaded into exosomes by direct incubation. Controllable removal of PEG could be achieved by breaking thioketal (TK) through reactive oxygen species (ROS), which was produced by Ce6 under ultrasound irradiation. The whole platform was called SmartExo. The stealth effects were analyzed in RAW264.7 cells and C57BL/6 mice via tracing the exosomes. To confirm the efficacy of the engineered smart exosomes, Bone morphogenetic protein 7 (Bmp7) mRNA was encapsulated into exosomes by transfection of overexpressing plasmid, followed by stealth coating, with the exosomes designated as SmartExo@Bmp7. Therapeutic advantages of SmartExo@Bmp7 were proved by targeted delivering Bmp7 mRNA to omental adipose tissue (OAT) of obese C57BL/6 mice for browning induction. SmartExo platform was successfully constructed without changing the basic characteristics of exosomes. The engineered exosomes effectively escaped from the phagocytosis by RAW264.7 and non-target organs. In addition, the SmartExo could be uptaken locally on-demand by ultrasound mediated removal of the stealth coat. Compared with control exosomes, SmartExo@Bmp7 effectively delivered Bmp7 mRNA into OAT upon ultrasound irradiation, and induced OAT browning, as evidenced by the histology of OAT and increased expression of uncoupling protein 1 (Ucp1). CONCLUSIONS: The proposed SmartExo-based delivery platform, which minimizes side effects and maximizing drug efficacy, offers a novel safe and efficient approach for targeted drug delivery. As a proof, the SmartExo@Bmp7 induced local white adipose tissue browning, and it would be a promising strategy for anti-obesity therapy.

Multifunctional Natural Polymer Nanoparticles as Antifibrotic Gene Carriers for CKD Therapy.

BACKGROUND: Progressive fibrosis is the underlying pathophysiological process of CKD, and targeted prevention or reversal of the profibrotic cell phenotype is an important goal in developing therapeutics for CKD. Nanoparticles offer new ways to deliver antifibrotic therapies to damaged tissues and resident cells to limit manifestation of the profibrotic phenotype. METHODS: We focused on delivering plasmid DNA expressing bone morphogenetic protein 7 (BMP7) or hepatocyte growth factor (HGF)-NK1 (HGF/NK1) by encapsulation within chitosan nanoparticles coated with hyaluronan, to safely administer multifunctional nanoparticles containing the plasmid DNA to the kidneys for localized and sustained expression of antifibrotic factors. We characterized and evaluated nanoparticles in vitro for biocompatibility and antifibrotic function. To assess antifibrotic activity in vivo, we used noninvasive delivery to unilateral ureteral obstruction mouse models of CKD. RESULTS: Synthesis of hyaluronan-coated chitosan nanoparticles containing plasmid DNA expressing either BMP7 or NGF/NKI resulted in consistently sized nanoparticles, which-following endocytosis driven by CD44+ cells-promoted cellular growth and inhibited fibrotic gene expression in vitro. Intravenous tail injection of these nanoparticles resulted in approximately 40%-45% of gene uptake in kidneys in vivo. The nanoparticles attenuated the development of fibrosis and rescued renal function in unilateral ureteral obstruction mouse models of CKD. Gene delivery of BMP7 reversed the progression of fibrosis and regenerated tubules, whereas delivery of HGF/NK1 halted CKD progression by eliminating collagen fiber deposition. CONCLUSIONS: Nanoparticle delivery of HGF/NK1 conveyed potent antifibrotic and proregenerative effects. Overall, this research provided the proof of concept on which to base future investigations for enhanced targeting and transfection of therapeutic genes to kidney tissues, and an avenue toward treatment of CKD.