Harnessing the Biomimetic Effect of Macromolecular Crowding in the Cell-Derived Model of Clubfoot Fibrosis.

Fibrotic changes in pediatric clubfoot provide an opportunity to improve corrective therapy and prevent relapses with targeted drugs. This study defines the parameters of clubfoot fibrosis and presents a unique analysis of a simple pseudo-3D in vitro model for disease-specific high-throughput drug screening experiments. The model combines clubfoot-derived fibroblasts with a biomimetic cultivation environment induced by the water-soluble polymers Ficoll and Polyvinylpyrrolidone, utilizing the principle of macromolecular crowding. We achieved higher conversion of soluble collagen into insoluble collagen, accelerated formation of the extracellular matrix layer and upregulated fibrosis-related genes in the mixed Ficoll environment. To test the model, we evaluated the effect of a potential antifibrotic drug, minoxidil, emphasizing collagen content and cross-linking. While the model amplified overall collagen deposition, minoxidil effectively blocked the expression of lysyl hydroxylases, which are responsible for the increased occurrence of specific collagen cross-linking in various fibrotic tissues. This limited the formation of collagen cross-link in both the model and control environments. Our findings provide a tool for expanding preclinical research for clubfoot and similar fibroproliferative conditions.

Increased Collagen Crosslinking in Stiff Clubfoot Tissue: Implications for the Improvement of Therapeutic Strategies.

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, ß-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10-40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

Integrated Bioinformatics Analysis Reveals Potential Pathway Biomarkers and Their Interactions for Clubfoot.

BACKGROUND Congenital talipes equinovarus (clubfoot), one of the most regular pediatric congenital skeletal anomalies, seriously affects the normal growth and development of about 1 in 1000 newborns. Although it has been investigated widely, the etiology and pathogenesis of clubfoot are still controversial. MATERIAL AND METHODS g: Profiler, NetworkAnalyst and WebGestalt were used to probe the enriched signaling pathways by using the Gene Ontology (GO), Human Phenotype Ontology (HP), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome (REAC), and WikiPathways (WP) databases. Large numbers of enriched signaling pathways were identified using the integrated bioinformatics enrichment analyses. RESULTS Apoptosis or programmed cell death (PCD), disease, muscle contraction, metabolism, and immune system were the top functions. Embryo or organ morphogenesis and development, cell or muscle contraction, and apoptosis were the top biological processes, and cell/muscle contraction and apoptosis were the top molecular functions using enriched GO terms analysis. There were a large number of complex interactions in the genes, enriched pathways, and transcription factor (TF)-miRNA co-regulatory networks. Transcription factors such as FOXN3, GLI3, HOX, and NCOR2 family regulated the gene expression of APAF1, BCL2, BID, CASP, MTHFR, and TPM family. CONCLUSIONS The results of bioinformatics enrichment analysis not only supported the previously proposed hypotheses, e.g., extracellular matrix (ECM) abnormality, fetal movement reducing, genetic abnormality, muscle abnormality, neurological abnormality, skeletal abnormality and vascular abnormality, but also indicated that cellular or immune responses to external stimulus, molecular transport and metabolism may be new etiological mechanisms in clubfoot.

Increased Microvessel and Arteriole Density in the Contracted Side of the Relapsed Clubfoot.

BACKGROUND: Clubfoot deformity (pes equinovarus) is one of the most common birth defects, and its etiology is still unknown. Initial clubfoot treatment is based on the Ponseti method throughout most of the world. Despite the effectiveness of this therapy, clubfoot may relapse. Recent studies confirm the theory of active fibrotic remodeling processes in the extracellular matrix of the affected tissue. The aim of this study was to clarify whether relapses in clubfoot therapy are associated with altered angiogenesis and to suggest possible regulatory pathways of this pathologic process. METHODS: We compared microvessel density, arteriole density, and concentration of angioproliferative-related proteins found between tissues in the contracted, that is, the medial side (M-side), and noncontracted, that is, the lateral side (L-side) of the relapsed clubfeet. Tissue samples from 10 patients were analyzed. Histopathologic analysis consisted of immunohistochemistry and image analysis. Real-time polymerase chain reaction was used to study mRNA expression. RESULTS: An increase in microvessel and arteriole density was noted in contracted, relapsed clubfoot tissue. This was accompanied by a significant increase in the levels of the vascular endothelial growth factor, vascular endothelial growth factor receptor 2, ß catenin and active ß catenin. Vascular endothelial growth factor, vascular endothelial growth factor receptor 2, and CD31 overexpression was also seen with mRNA analysis. CONCLUSIONS: Increased microvessel and arteriole density in the contracted side of the relapsed clubfoot was noted. These processes are mediated by specific proangiogenic proteins that are overexpressed in the contracted tissue. These findings contribute to the etiology and the development of relapses in the treatment of clubfoot. LEVEL OF EVIDENCE: Level II-analytical and prospective.

EIF4A3 deficient human iPSCs and mouse models demonstrate neural crest defects that underlie Richieri-Costa-Pereira syndrome.

Biallelic loss-of-function mutations in the RNA-binding protein EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS), an autosomal recessive condition mainly characterized by craniofacial and limb malformations. However, the pathogenic cellular mechanisms responsible for this syndrome are entirely unknown. Here, we used two complementary approaches, patient-derived induced pluripotent stem cells (iPSCs) and conditional Eif4a3 mouse models, to demonstrate that defective neural crest cell (NCC) development explains RCPS craniofacial abnormalities. RCPS iNCCs have decreased migratory capacity, a distinct phenotype relative to other craniofacial disorders. Eif4a3 haploinsufficient embryos presented altered mandibular process fusion and micrognathia, thus recapitulating the most penetrant phenotypes of the syndrome. These defects were evident in either ubiquitous or NCC-specific Eif4a3 haploinsufficient animals, demonstrating an autonomous requirement of Eif4a3 in NCCs. Notably, RCPS NCC-derived mesenchymal stem-like cells (nMSCs) showed premature bone differentiation, a phenotype paralleled by premature clavicle ossification in Eif4a3 haploinsufficient embryos. Likewise, nMSCs presented compromised in vitro chondrogenesis, and Meckel's cartilage was underdeveloped in vivo. These findings indicate novel and essential requirements of EIF4A3 for NCC migration and osteochondrogenic differentiation during craniofacial development. Altogether, complementary use of iPSCs and mouse models pinpoint unique cellular mechanisms by which EIF4A3 mutation causes RCPS, and provide a paradigm to study craniofacial disorders.

Retinoic Acid Promotes Retinoic Acid Signaling by Suppression of Pitx1 In Tendon Cells: A Possible Mechanism of a Clubfoot-Like Phenotype Induced by Retinoic Acid.

BACKGROUND The pathogenesis of idiopathic congenital clubfoot (CCF) is unknown. Although some familial patients have Pitx1 mutations, and the Pitx1+/- genotype causes a clubfoot-like phenotype in mice, the mechanism of Pitx1-induced CCF is unknown. MATERIAL AND METHODS We used tibialis anterior tendon samples to detect the expression of Pitx1 in idiopathic and neurogenic clubfoot patients. After obtaining Sprague-Dawley (SD) rat Achilles tendon cells, the expression of Pitx1 was knocked down by SiRNA. After 48 h of culture, mass spectrometry was used to quantitatively analyze proteins. Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to assess the downstream pathway of PITX1. The relationship between Pitx1 and the promoter region of deacetylase 1 (Sirtuin-1 and Sirt1) was examined by luciferase and ChIP assays. RESULTS We found that Pitx1 expression in the tendon samples of idiopathic CCF patients was downregulated. Mass spectrometry analysis revealed that the inhibition of Pitx1 induced the downregulation of Sirt1 expression in tendon cells. Luciferase and ChIP assays confirmed that Pitx1 binds to the promoter region of SIRT1 and promotes Sirt1 gene transcription. Further results showed that, after the inhibition of Pitx1 in tendon cells, CRABP2 acetylation increased, the nuclear import of CRABP2 was enhanced, and the expression of RARß2 increased. After the inhibition of Pitx1, RARß2 expression was further increased by RA treatment in tendon cells. In the presence of retinoic acid, the expression of Pitx1 was inhibited in tendon cells. CONCLUSIONS Pitx1 binds to the promoter region of SIRT1 and promotes the transcription of SIRT1. Positive feedback occurs between RA signaling and Pitx1.

RBM10: Harmful or helpful-many factors to consider.

RBM10 is an RNA binding motif (RBM) protein expressed in most, if not all, human and animal cells. Interest in RBM10 is rapidly increasing and its clinical importance is highlighted by its identification as the causative agent of TARP syndrome, a developmental condition that significantly impacts affected children. RBM10's cellular functions are beginning to be explored, with initial studies demonstrating a tumor suppressor role. Very recently, however, contradictory results have emerged, suggesting a tumor promoter role for RBM10. In this review, we describe the current state of knowledge on RBM10, and address this dichotomy in RBM10 function. Furthermore, we discuss what may be regulating RBM10 function, particularly the importance of RBM10 alternative splicing, and the relationship between RBM10 and its paralogue, RBM5. As RBM10-related work is gaining momentum, it is critical that the various aspects of RBM10 molecular biology revealed by recent studies be considered moving forward. It is only if these recent advances in RBM10 structure and function are considered that a clearer insight into RBM10 function, and the disease states with which RBM10 mutation is associated, will be gained.

A Novel Splice-Site Mutation in VEGFC Is Associated with Congenital Primary Lymphoedema of Gordon.

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area.

Functional Assessment of Clubfoot Associated HOXA9, TPM1, and TPM2 Variants Suggests a Potential Gene Regulation Mechanism.

BACKGROUND: Isolated nonsyndromic clubfoot is a common birth defect affecting 135,000 newborns worldwide each year. Although treatment has improved, substantial long-term morbidity persists. Genetic causes have been implicated in family-based studies but the genetic changes have eluded identification. Previously, using a candidate gene approach in our family-based dataset, we identified associations between clubfoot and four single nucleotide polymorphisms (SNPs) located in potential regulatory regions of genes involved in muscle development and patterning (HOXA9) and muscle function (TPM1 and TPM2) were identified. QUESTIONS/PURPOSES: Four SNPs, rs3801776/HOXA9, rs4075583/TPM1, rs2025126/TPM2, and rs2145925/TPM2, located in potential regulatory regions, were evaluated to determine whether they altered promoter activity. METHODS: Electrophoretic mobility shift assays were performed on these four SNPs to identify allele-specific DNA-protein interactions. SNPs showing differential banding patterns were assessed for effect on promoter activity by luciferase assay. Undifferentiated (for HOXA9) and differentiated (for TPM1 and TPM2) mouse cells were used in functional assays as a proxy for the in vivo developmental stage. RESULTS: Functional analyses showed that the ancestral alleles of rs3801776/HOXA9, rs4075583/TPM1, and rs2025126/TPM2 and the alternate allele of rs2145925/TPM2 created allele-specific nuclear protein interactions and caused higher promoter activity. Interestingly, while rs4075583/TPM1 showed an allele-specific nuclear protein interaction, an effect on promoter activity was observed only when rs4075583/TPM1 was expressed in the 1.7kb haplotype construct. CONCLUSION: Our results show that associated promoter variants in HOXA9, TPM1, and TPM2, alter promoter expression suggesting that they have a functional role. Moreover and importantly, we show that alterations in promoter activity may be observed only in the context of the genomic architecture. Therefore, future studies focusing on proteins binding to these regulatory SNPs may provide important key insights into gene regulation in clubfoot. CLINICAL RELEVANCE: Identifying the genetic risk signature for clubfoot is important to provide accurate genetic counseling for at-risk families, for development of prevention programs and new treatments.

A review of craniofacial disorders caused by spliceosomal defects.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders.

Proteomic analysis of the extracellular matrix in idiopathic pes equinovarus.

Idiopathic pes equinovarus is a congenital deformity of the foot and lower leg defined as a fixation of the foot in adduction, supination, and varus. Although the pathogenesis of clubfoot remains unclear, it has been suggested that fibroblasts and growth factors are involved. To directly analyze the protein composition of the extracellular matrix in contracted tissue of patients with clubfoot. A total of 13 infants with idiopathic clubfoot treated with the Ponseti method were included in the present study. Tissue samples were obtained from patients undergoing surgery for relapsed clubfeet. Contracted tissues were obtained from the medial aspect of the talonavicular joint. Protein was extracted after digestion and delipidation using zip-tip C18. Individual collagenous fractions were detected using a chemiluminescent assay. Amino acid analysis of tissue samples revealed a predominance of collagens, namely collagen types I, III, and VI. The high content of glycine and h-proline suggests a predominance of collagens I and III. A total of 19 extracellular matrix proteins were identified. The major result of the present study was the observation that the extracellular matrix in clubfoot is composed of an additional 16 proteins, including collagens V, VI, and XII, as well as the previously described collagen types I and III and transforming growth factor ß. The characterization of the general protein composition of the extracellular matrix in various regions of clubfoot may help in understanding the pathogenesis of this anomaly and, thus, contribute to the development of more efficacious therapeutic approaches.

Pitx1 haploinsufficiency causes clubfoot in humans and a clubfoot-like phenotype in mice.

Clubfoot affects 1 in 1000 live births, although little is known about its genetic or developmental basis. We recently identified a missense mutation in the PITX1 bicoid homeodomain transcription factor in a family with a spectrum of lower extremity abnormalities, including clubfoot. Because the E130K mutation reduced PITX1 activity, we hypothesized that PITX1 haploinsufficiency could also cause clubfoot. Using copy number analysis, we identified a 241 kb chromosome 5q31 microdeletion involving PITX1 in a patient with isolated familial clubfoot. The PITX1 deletion segregated with autosomal dominant clubfoot over three generations. To study the role of PITX1 haploinsufficiency in clubfoot pathogenesis, we began to breed Pitx1 knockout mice. Although Pitx1(+/-) mice were previously reported to be normal, clubfoot was observed in 20 of 225 Pitx1(+/-) mice, resulting in an 8.9% penetrance. Clubfoot was unilateral in 16 of the 20 affected Pitx1(+/-) mice, with the right and left limbs equally affected, in contrast to right-sided predominant hindlimb abnormalities previously noted with complete loss of Pitx1. Peroneal artery hypoplasia occurred in the clubfoot limb and corresponded spatially with small lateral muscle compartments. Tibial and fibular bone volumes were also reduced. Skeletal muscle gene expression was significantly reduced in Pitx1(-/-) E12.5 hindlimb buds compared with the wild-type, suggesting that muscle hypoplasia was due to abnormal early muscle development and not disuse atrophy. Our morphological data suggest that PITX1 haploinsufficiency may cause a developmental field defect preferentially affecting the lateral lower leg, a theory that accounts for similar findings in human clubfoot.

[Possible role of GLI3 gene in the pathogenesis of idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV). METHOD: Potential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR. Following generation of rat model for ICTEV, mRNA and protein levels of GLI3 were evaluated by real-time PCR and immunohistochemistry and Western blotting. RESULTS: No mutation was found in exons 1 - 8 and 13 of GLI3 gene among the 84 ICTEV patients. No expression of GLI3 gene was detected in the flexor hallucis longus of ICTEV patients or normal controls. Expression of Gli3, in terms of both mRNA and protein, was stronger in the hindlimb of ICTEV rat embryos compared with normal controls. CONCLUSION: Mutation in the coding region of GLI3 may not be responsible for the occurrence of ICTEV. However, there may still be connection between abnormal expression of the gene and pathogenesis of ICTEV.

A preliminary study pointing out the role of serum prolidase activity and oxidative-antioxidative status parameters during the treatment process of patients with idiopathic clubfoot.

BACKGROUND: We aimed to investigate serum prolidase activity and to find out its association with oxidative-antioxidative status in patients with idiopathic clubfoot and during the course of the disease. MATERIAL AND METHODS: Oxidative status parameters, including total free sulfhydryl groups (-SH), total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI), as well as serum prolidase activity were assessed at the beginning of the treatment in patients with idiopathic clubfoot (n = 38), at the end of 3 months during the treatment of the disease and in healthy controls (n = 40). All patients were managed with the Ponseti method and severity of the foot deformity was evaluated according to the Pirani Severity Score. RESULTS: Serum prolidase activity, TOS and OSI values of the patients at the beginning of the treatment were found to be significantly higher but -SH and TAC values were found to be significantly lower as compared to controls. In the treatment process, a significant decrease in serum prolidase activity, TOS and OSI values and Pirani Severity Score of the patients was observed, however a significant increase in -SH and TAC values of the patients was observed at the end of 3 months during the treatment of the disease as compared to the beginning of the treatment. CONCLUSION: Elevated levels of serum prolidase activity, TOS and OSI, and decreased levels of -SH and TAC may be associated with idiopathic clubfoot, and that these parameters may be useful adjunctive tools for follow-up in patients with idiopathic clubfoot.

Maternal smoking during pregnancy aggravated muscle phenotype in FHL1-/y offspring mice similar to congenital clubfoot through P2RX7-mediated pyroptosis.

Congenital clubfoot (CCF) is a common birth defect. Maternal smoking during pregnancy increases the risk of CCF. In previous research, we found muscle phenotypes similar to CCF in four and a half LIM domain protein 1 (FHLI) offspring mice (FHL1-/y). However, the role of P2RX7-mediated pyroptosis in the effect of cigarette smoke (CS) on the skeletal muscle of FHL1-/y mice during pregnancy is unclear. In the present study, pregnant mice at 11 days of gestation were exposed to CS and male offspring of wild-type (WT) and FHL1-/y mice were divided into four groups (Control-WT, Control-KO, CS-WT, CS-KO). The histomorphology of lower limb muscles was examined using hematoxylin and eosin (H&E) staining. P2RX7, indicators of pyroptosis (NLRP3, ASC, cleaved-caspase 1, IL-1ß), and cytoskeletal proteins (MYBPC2, LDB3) were also detected using immunoblotting. CS exposure during pregnancy aggravated the muscle phenotype similar to CCF in FHL1-/y offspring mice. FHL1 gene knockout (KO) or CS exposure during pregnancy each activated the expression of P2RX7, cell pyroptosis-related proteins (NLRP3, ASC, cleaved-caspase 1, IL-1ß), a muscle injury marker (MYOD1), and cytoskeletal proteins (MYBPC2, LDB3); these two factors had an additive effect. The results showed maternal smoking during pregnancy aggravated muscle phenotype similar to CCF in FHL1-/y offspring mice through P2RX7-mediated pyroptosis.

Beta-catenin mediates soft tissue contracture in clubfoot.

The contracted tissues from clubfeet resemble tissues from other fibroproliferative disorders such as palmar fibromatosis. Beta-catenin-mediated signaling is a crucial pathway controlling the fibroproliferative response in many fibroproliferative disorders. To determine if beta-catenin signaling plays a role in clubfoot, contracted and less contracted tissues from clubfeet were studied using Western analysis to determine the protein level of beta-catenin. Primary cell cultures were established from these tissues, and they were treated with either lithium to increase beta-catenin or Dickkopf-1 to inhibit beta-catenin. RNA was extracted from the cells and analyzed to determine how beta-catenin regulates expression of Type III collagen, an extracellular matrix protein upregulated in contracted clubfoot tissue. There was a more than twofold increase in beta-catenin protein in the contracted tissues. Treatment with either lithium or Dickkopf-1 showed Type III collagen RNA expression positively correlated with the protein level of beta-catenin. These data support the concept that beta-catenin-mediated signaling plays an important role regulating contracture in clubfeet. Because pharmacologic agents are under development to block this signaling pathway, such drugs could be used in cases of severe stiffness to improve range of motion or to decrease the need for radical surgical approaches.

The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus.

BACKGROUND: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development. METHODS: Using the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3. RESULTS: Gli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative Hoxd13 binding sites. Using EMSA, the Hoxd13 binding site 2 was found to directly interact with Hoxd13 in vitro. ChIP assays of the Hoxd13-Gli3 promoter complex from a developing limb confirmed that endogenous Hoxd13 interacts with this region in vivo. CONCLUSION: Our findings suggest that HoxD13 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HoxD13.

[The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus].

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.

Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins.

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Monogenic Forms of Hypertension.

Essential hypertension is a highly prevalent disease in the general population. Secondary hypertension is characterized by a specific and potentially reversible cause of increased blood pressure levels. Some secondary endocrine forms of hypertension are common (caused by uncontrolled cortisol, aldosterone, or catecholamines production). This article describes rare monogenic forms of hypertension, characterized by electrolyte disorders and suppressed renin-aldosterone axis. They represent simple models for the physiology of renal control of sodium levels and plasma volume, thus reaching a high scientific interest. Furthermore, they could explain some features closer to the essential phenotype of hypertension, suggesting a mechanistically driven personalized treatment.