RESUMO
AIM: To determine the dose-response relationship of collagenase Clostridium histolyticum (CCH) on collagen content and the change in muscle fiber bundle stiffness after ex vivo treatment of adductor longus biopsies with CCH in children with cerebral palsy (CP). METHOD: Biopsy samples of adductor longus from children with CP (classified in Gross Motor Function Classification System levels IV and V) were treated with 0 U/mL, 200 U/mL, 350 U/mL, or 500 U/mL CCH; percentage collagen reduction was measured to determine the dose-response. Peak and steady-state stresses were determined at 1%, 2.5%, 5%, and 7.5% strain increments; Young's modulus was calculated. RESULTS: Eleven patients were enrolled (nine males, two females, mean age at surgery 6 years 5 months; range: 2-16 years). A linear CCH dose-response relationship was determined. Peak and steady-state stress generation increased linearly at 5.9/2.3mN/mm2 , 12.4/5.3mN/mm2 , 22.2/9.7mN/mm2 , and 33.3/15.5mN/mm2 at each percentage strain increment respectively. After CCH treatment, peak and steady-state stress generation decreased to 3.2/1.2mN/mm2 , 6.5/2.9mN/mm2 , 12.2/5.7mN/mm2 , and 15.4/7.7mN/mm2 respectively (p < 0.004). Young's modulus decreased from 205 kPa to 100 kPa after CCH (p = 0.003). INTERPRETATION: This preclinical ex vivo study provides proof of concept for the use of collagenase to decrease muscle stiffness in individuals with CP.
Assuntos
Paralisia Cerebral , Masculino , Criança , Feminino , Humanos , Colagenase Microbiana/uso terapêutico , Músculo Esquelético , Colágeno , Fibras Musculares Esqueléticas , Resultado do TratamentoRESUMO
AIM: To analyze transcriptomes from muscle tissue and cells to improve our understanding of differences in gene expression and molecular function in cerebral palsy (CP) muscle. METHOD: In this case-control study, eight participants with CP (five males, three females; mean [SD] age 14y 2mo [1y 8mo]) and 11 comparison individuals (eight males, three females; mean [SD] age 14y 0mo [2y 6mo]) were enrolled after informed consent/assent and skeletal muscle was obtained during surgery. RNA was extracted from tissue and from primary satellite cells grown to form myotubes in vitro. RNA sequencing data were analyzed using validated informatics pipelines. RESULTS: Analysis identified expression of 6308 genes in the tissue samples and 7459 in the cultured cells. Significant differential expression between CP and control was identified in 87 genes in the tissue and 90 genes in isolated satellite cell-derived myotube cultures. INTERPRETATION: Both tissue and cell analyses identified differential expression of genes associated with muscle development and multiple pathways of interest. What this paper adds Expression differences were found in muscle tissue and in isolated muscle cells. There was low variability in expression among cells isolated from different muscles. Expression differences suggest complex functional alterations in spastic cerebral palsy.
Assuntos
Paralisia Cerebral/genética , Espasticidade Muscular/genética , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Adolescente , Estudos de Casos e Controles , Criança , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , RNA-Seq , TranscriptomaRESUMO
BACKGROUND: Spastic cerebral palsy (CP) is a leading cause of physical disability. Most people with spastic CP are born with it, but early diagnosis is challenging, and no current biomarker platform readily identifies affected individuals. The aim of this study was to evaluate epigenetic profiles as biomarkers for spastic CP. A novel analysis pipeline was employed to assess DNA methylation patterns between peripheral blood cells of adolescent subjects (14.9 ± 0.3 years old) with spastic CP and controls at single CpG site resolution. RESULTS: Significantly hypo- and hyper-methylated CpG sites associated with spastic CP were identified. Nonmetric multidimensional scaling fully discriminated the CP group from the controls. Machine learning based classification modeling indicated a high potential for a diagnostic model, and 252 sets of 40 or fewer CpG sites achieved near-perfect accuracy within our adolescent cohorts. A pilot test on significantly younger subjects (4.0 ± 1.5 years old) identified subjects with 73% accuracy. CONCLUSIONS: Adolescent patients with spastic CP can be distinguished from a non-CP cohort based on DNA methylation patterns in peripheral blood cells. A clinical diagnostic test utilizing a panel of CpG sites may be possible using a simulated classification model. A pilot validation test on patients that were more than 10 years younger than the main adolescent cohorts indicated that distinguishing methylation patterns are present earlier in life. This study is the first to report an epigenetic assay capable of distinguishing a CP cohort.
Assuntos
Biomarcadores/análise , Paralisia Cerebral/diagnóstico , Paralisia Cerebral/genética , Metilação de DNA , Epigenômica , Aprendizado de Máquina , Reconhecimento Automatizado de Padrão , Análise de Sequência de DNA/métodos , Adolescente , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genoma Humano , Humanos , MasculinoRESUMO
STUDY HYPOTHESIS: Susceptibility to inherited cryptorchidism in the LE/orl rat may be associated with genetic loci that influence developmental patterning of the gubernaculum by the fetal testis. STUDY FINDING: Cryptorchidism in the LE/orl rat is associated with a unique combination of homozygous minor alleles at multiple loci, and the encoded proteins are co-localized with androgen receptor (AR) and Leydig cells in fetal gubernaculum and testis, respectively. WHAT IS KNOWN ALREADY: Prior studies have shown aberrant perinatal gubernacular migration, muscle patterning defects and reduced fetal testicular testosterone in the LE/orl strain. In addition, altered expression of androgen-responsive, cytoskeletal and muscle-related transcripts in the LE/orl fetal gubernaculum suggest a role for defective AR signaling in cryptorchidism susceptibility. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The long-term LE/orl colony and short-term colonies of outbred Crl:LE and Crl:SD, and inbred WKY/Ncrl rats were maintained for studies. Animals were intercrossed (LE/orl X WKY/Ncrl), and obligate heterozygotes were reciprocally backcrossed to LE/orl rats to generate 54 F2 males used for genotyping and/or linkage analysis. At least five fetuses per gestational time point from two or more litters were used for quantitative real-time RT-PCR (qRT-PCR) and freshly harvested embryonic (E) day 17 gubernaculum was used to generate conditionally immortalized cell lines. We completed genotyping and gene expression analyses using genome-wide microsatellite markers and single nucleotide polymorphism (SNP) arrays, PCR amplification, direct sequencing, restriction enzyme digest with fragment analysis, whole genome sequencing (WGS), and qRT-PCR. Linkage analysis was performed in Haploview with multiple testing correction, and qRT-PCR data were analyzed using ANOVA after log transformation. Imaging was performed using custom and commercial antibodies directed at candidate proteins in gubernaculum and testis tissues, and gubernaculum cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: LE/orl rats showed reduced fertility and fecundity, and higher risk of perinatal death as compared with Crl:LE rats, but there were no differences in breeding outcomes between normal and unilaterally cryptorchid males. Linkage analysis identified multiple peaks, and with selective breeding of outbred Crl:LE and Crl:SD strains for alleles within two of the most significant (P < 0.003) peaks on chromosomes 6 and 16, we were able to generate a non-LE/orl cryptorchid rat. Associated loci contain potentially functional minor alleles (0.25-0.36 in tested rat strains) including an exonic deletion in Syne2, a large intronic insertion in Ncoa4 (an AR coactivator) and potentially deleterious variants in Solh/Capn15, Ankrd28, and Hsd17b2. Existing WGS data indicate that homozygosity for these combined alleles does not occur in any other sequenced rat strain. We observed a modifying effect of the Syne2(del) allele on expression of other candidate genes, particularly Ncoa4, and for muscle and hormone-responsive transcripts. The selected candidate genes/proteins are highly expressed, androgen-responsive and/or co-localized with developing muscle and AR in fetal gubernaculum, and co-localized with Leydig cells in fetal testis. LIMITATIONS, REASONS FOR CAUTION: The present study identified multiple cryptorchidism-associated linkage peaks in the LE/orl rat, containing potentially causal alleles. These are strong candidate susceptibility loci, but further studies are needed to demonstrate functional relevance to the phenotype. WIDER IMPLICATIONS OF THE FINDINGS: Association data from both human and rat models of spontaneous, nonsyndromic cryptorchidism support a polygenic etiology of the disease. Both the present study and a human genome-wide association study suggest that common variants with weak effects contribute to susceptibility, and may exist in genes encoding proteins that participate in AR signaling in the developing gubernaculum. These findings have potential implications for the gene-environment interaction in the etiology of cryptorchidism. LARGE SCALE DATA: Sequences were deposited in the Rat Genome Database (RGD, http://rgd.mcw.edu/). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by: R01HD060769 from the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), 2P20GM103446 and P20GM103464 from the National Institute of General Medical Sciences (NIGMS), and Nemours Biomedical Research. The authors have no competing interests to declare.
Assuntos
Criptorquidismo/veterinária , Herança Multifatorial , Ratos Long-Evans/genética , Doenças dos Roedores/genética , Alelos , Androgênios/fisiologia , Animais , Criptorquidismo/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Ligação Genética , Predisposição Genética para Doença , Infertilidade Masculina/genética , Infertilidade Masculina/veterinária , Células Intersticiais do Testículo/metabolismo , Masculino , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/fisiologia , Ratos , Ratos Endogâmicos WKY , Ratos Mutantes , Reação em Cadeia da Polimerase em Tempo Real , Testículo/embriologiaRESUMO
PURPOSE: Gubernaculum-cremaster complex development is hormonally regulated and abnormal in a cryptorchid rat model. Using cell tracking techniques and imaging we studied myogenic phenotypes and fates in the fetal rat gubernaculum-cremaster complex. MATERIALS AND METHODS: Embryonic day 17 gubernaculum-cremaster complexes were labeled with CellTracker™ or the DNA synthesis marker EdU (5-ethynyl-2'-deoxyuridine), or immobilized in Matrigel® and grown in culture. Embryonic day 17 to 21 gubernaculum-cremaster complex sections and cells were imaged using wide field and deconvolution immunofluorescence microscopy, and muscle and/or myofibroblast specific antibodies. Deconvolved image stacks were used to create a 3-dimensional model of embryonic day 21 gubernaculum-cremaster complex muscle. RESULTS: PAX7 (paired box 7) positive and myogenin positive muscle precursors were visible in a desmin-rich myogenic zone between muscle layers that elongated and became thicker during development. Gubernaculum-cremaster complex inner mesenchymal cells expressed desmin and αSMA (α smooth muscle actin) at lower levels than in the myogenic zone. After pulse labeling with CellTracker or EdU mesenchymal cells became incorporated into differentiated muscle. Conversely, mesenchymal cells migrated beyond Matrigel immobilized gubernaculum-cremaster complexes, expressed PAX7 and fused to form striated myotubes. Mesenchymal gubernaculum-cremaster complex cell lines proliferated more than 40 passages and showed contractile behavior but did not form striated muscle. Our 3-dimensional gubernaculum-cremaster complex model had 2 orthogonal ventral layers and an arcing inner layer of muscle. CONCLUSIONS: Our data suggest that mesenchymal cells in the peripheral myogenic zone of the fetal gubernaculum-cremaster complex contribute to formation of a distinctively patterned cremaster muscle. Nonmyogenic, desmin and αSMA positive gubernaculum-cremaster complex mesenchymal cells proliferate and have a myofibroblast-like phenotype in culture. Intrinsic mechanical properties of these divergent cell types may facilitate perinatal inversion of the gubernaculum-cremaster complex.
Assuntos
Músculos Abdominais/embriologia , Diferenciação Celular/fisiologia , Gubernáculo/embriologia , Células-Tronco Mesenquimais/fisiologia , Desenvolvimento Muscular/fisiologia , Miofibroblastos/fisiologia , Fenótipo , Músculos Abdominais/citologia , Animais , Linhagem Celular , Gubernáculo/citologia , Ratos , Ratos Long-EvansRESUMO
Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility; expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multiarm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multiarm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and cross-linking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, microheterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds.
Assuntos
Hidrogéis/química , Proteínas de Insetos/química , Polietilenoglicóis/química , Polímeros/química , Alicerces Teciduais/química , Elasticidade , Elastina/química , Elastômeros , Humanos , Hidrogéis/administração & dosagem , Proteínas de Insetos/administração & dosagem , Metaloproteinases da Matriz/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Peptídeos/química , Polietilenoglicóis/administração & dosagem , Polímeros/administração & dosagem , Reologia/métodos , Engenharia Tecidual/métodosRESUMO
PURPOSE: Based on a genome-wide association study of testicular dysgenesis syndrome showing a possible association with TGFBR3, we analyzed data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal. MATERIALS AND METHODS: We excluded samples based on strict quality control criteria, leaving 844 cases and 2,718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform (ie Illumina® HumanHap550, version 1 or 3, or Human610-Quad, version 1 BeadChip in group 1 and Human OmniExpress 12, version 1 BeadChip platform in group 2). Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of data for groups 1 and 2, and selective genotyping of independent cases (330) and controls (324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum. RESULTS: We identified suggestive (p ≤ 1× 10(-4)) association of markers in/near TGFBR3, including rs9661103 (OR 1.40; 95% CI 1.20, 1.64; p = 2.71 × 10(-5)) and rs10782968 (OR 1.58; 95% CI 1.26, 1.98; p = 9.36 × 10(-5)) in groups 1 and 2, respectively. In subgroup analyses we observed strongest association of rs17576372 (OR 1.42; 95% CI 1.24, 1.60; p = 1.67 × 10(-4)) with proximal and rs11165059 (OR 1.32; 95% CI 1.15, 1.38; p = 9.42 × 10(-4)) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior genome-wide association study signal (rs12082710) was marginal (OR 1.13; 95% CI 0.99, 1.28; p = 0.09 for group 1), and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild-type and cryptorchid rat fetal gubernaculum. CONCLUSIONS: These data suggest complex or phenotype specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFß signaling.
Assuntos
Criptorquidismo/genética , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Criança , Pré-Escolar , Humanos , Lactente , Masculino , FenótipoRESUMO
Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.
Assuntos
Criptorquidismo/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Desenvolvimento Muscular/fisiologia , Transdução de Sinais/fisiologia , Animais , Sequência de Bases , DNA/genética , Feminino , Desenvolvimento Fetal/fisiologia , Variação Genética , Genótipo , Insulina , Masculino , Desenvolvimento Muscular/genética , Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores Acoplados a Proteínas G , Testículo/crescimento & desenvolvimentoRESUMO
BACKGROUND: Higher neighborhood deprivation is associated with hypertension diagnosis in youth. In this study, we assess if there is an association between neighborhood deprivation and antihypertensive therapy prescription among insured youth with a primary hypertension diagnosis. METHODS: Using a retrospective cross-sectional design, we assessed the proportion of youth with a diagnosis of primary hypertension prescribed antihypertensive therapy. We evaluated the proportion of youth prescribed antihypertensive therapy and compared prescribing patterns by area deprivation index (ADI), age, sex, obesity diagnosis, race, ethnicity, and duration of Medicaid coverage. RESULTS: Of the 65,452 non-pregnant Delaware Medicaid recipients, 8-18 years of age, 1,145 (1.7%) had an International classification of diseases (ICD)-9/ICD-10 diagnosis of primary hypertension; 165 of the 1,145 (14%) were prescribed antihypertensive therapy. Factors associated with a greater odds of prescription by multivariable logistic regression were age, obesity diagnosis, and duration of Medicaid full benefit coverage. Odds of antihypertensive therapy prescription did not vary by race, ethnicity, or ADI. CONCLUSIONS: Antihypertensive therapy prescription rates are poor despite national guideline recommendations. Among youth receiving Delaware Medicaid between 2014 and 2019, prescription proportions were highest among youth of older age, with an obesity diagnosis, and among youth with longer duration of Medicaid benefit coverage. Although high area deprivation has been shown to be associated with the diagnosis of hypertension, high vs. low area deprivation was not associated with greater antihypertensive therapy prescription among youth with primary hypertension. Our finding of a mismatch between hypertension diagnosis and antihypertensive therapy prescription highlights a potential disparity in antihypertensive therapy prescription in youth.
Assuntos
Anti-Hipertensivos , Hipertensão , Estados Unidos/epidemiologia , Adolescente , Humanos , Anti-Hipertensivos/uso terapêutico , Medicaid , Estudos Retrospectivos , Estudos Transversais , Delaware/epidemiologia , Hipertensão/diagnóstico , Hipertensão/tratamento farmacológico , Hipertensão/epidemiologia , Prescrições , Obesidade/tratamento farmacológico , Hipertensão Essencial/tratamento farmacológicoRESUMO
BACKGROUND: Wall stress is a useful concept to understand the progression of ventricular remodeling. We measured cumulative LV wall stress throughout the cardiac cycle over unit time and tested whether this "integrated wall stress (IWS)" would provide a reliable marker of total ventricular workload. METHODS AND RESULTS: We applied IWS to mice after experimental myocardial infarction (MI) and sham-operated mice, both at rest and under dobutamine stimulation. Small infarcts were created so as not to cause subsequent overt hemodynamic decompensation. IWS was calculated over one minute through simultaneous measurement of LV internal diameter and wall thickness by echocardiography and LV pressure by LV catheterization. At rest, the MI group showed concentric LV hypertrophy pattern with preserved LV cavity size, LV systolic function, and IWS comparable with the sham group. Dobutamine stimulation induced a dose-dependent increase in IWS in MI mice, but not in sham mice; MI mice mainly increased heart rate, whereas sham mice increased LV systolic and diastolic function. IWS showed good correlation with a product of peak-systolic wall stress and heart rate. We postulate that this increase in IWS in post-MI mice represents limited myocardial contractile reserve. CONCLUSION: We hereby propose that IWS provides a useful estimate of total ventricular workload in the mouse model and that increased IWS indicates limited LV myocardial contractile reserve.
Assuntos
Ventrículos do Coração/patologia , Contração Miocárdica/fisiologia , Remodelação Ventricular/fisiologia , Animais , Vasos Coronários/patologia , Dobutamina/farmacologia , Ecocardiografia , Frequência Cardíaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miocárdio/patologia , Oxigênio/metabolismo , Estresse MecânicoRESUMO
The Escobar variant of multiple pterygium syndrome (OMIM #265000) is a rare, autosomal recessive disorder associated with mutations in the γ-subunit of the nicotinic acetylcholine receptor (CHRNG). CHRNG is expressed in fetal muscle during motor development and contributes to the formation of neuromuscular junctions (NMJs). Anomalies in NMJ structure and function have not been investigated in patients with Escobar syndrome. We report five patients identified as having Escobar syndrome, from four families. In three families, the same mutation (c.459dupA) was identified in CHRNG. A biopsy from brachioradialis muscle was collected from a patient from one of these families and analyzed for NMJ organization using fluorescence microscopy. Compared to spinalis muscle from control patients with idiopathic scoliosis or cerebral palsy (CP), the patient with Escobar syndrome had a significantly higher degree of acetylcholine receptor present outside acetylcholinesterase and significantly less acetylcholinesterase outside acetylcholine receptors. Given the role of the acetylcholine receptor γ-subunit in fetal neuromuscular signal transduction and in establishing the primary encounter of muscle and motor nerve terminal, the CHRNG mutations described in Escobar syndrome may cause a broader disruption of postsynaptic proteins and result in aberrant development of the NMJ due to impaired prenatal neuromuscular transmission and/or abnormal neuromuscular synaptogenesis.
Assuntos
Anormalidades Múltiplas/genética , Hipertermia Maligna/genética , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/genética , Anormalidades da Pele/genética , Sinapses/patologia , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/fisiopatologia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Hipertermia Maligna/metabolismo , Hipertermia Maligna/fisiopatologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Escoliose/genética , Escoliose/metabolismo , Escoliose/fisiopatologia , Anormalidades da Pele/metabolismo , Anormalidades da Pele/fisiopatologia , Sinapses/metabolismoRESUMO
CircRNAs are a category of regulatory RNAs that have garnered significant attention in the field of regulatory RNA research due to their structural stability and tissue-specific expression. Their circular configuration, formed via back-splicing, results in a covalently closed structure that exhibits greater resistance to exonucleases compared to linear RNAs. The distinctive regulation of circRNAs is closely associated with several physiological processes, as well as the advancement of pathophysiological processes in several human diseases. Despite a good understanding of the biogenesis of circular RNA, details of their biological roles are still being explored. With the steady rise in the number of investigations being carried out regarding the involvement of circRNAs in various regulatory pathways, understanding the biological and clinical relevance of circRNA-mediated regulation has become challenging. Given the vast landscape of circRNA research in the development of the heart and vasculature, we evaluated cardiovascular system research as a model to critically review the state-of-the-art understanding of the biologically relevant functions of circRNAs. We conclude the review with a discussion of the limitations of current functional studies and provide potential solutions by which these limitations can be addressed to identify and validate the meaningful and impactful functions of circRNAs in different physiological processes and diseases.
Assuntos
Sistema Cardiovascular , RNA Circular , Humanos , RNA Circular/genética , RNA/genética , Biomarcadores , CoraçãoRESUMO
Institutional Development Award (IDeA) programs build research infrastructure in regions with historically low access to NIH funds. The Mentored Research Development Award (MRDA), a professional development program embedded in our IDeA-funded center, provides junior investigators with mentorship and effort offset to write a grant. We evaluated outcomes from the first eight years (2013-2021; N = 55) using administrative records, publicly available data, and a self-report survey (n = 46, 84% response rate). Fifteen MRDA recipients (27%) went on to receive NIH funding. Providing just-in-time grant-writing support may launch early career clinician-scientists in an IDeA state context.
RESUMO
Importance: The association between degree of neighborhood deprivation and primary hypertension diagnosis in youth remains understudied. Objective: To assess the association between neighborhood measures of deprivation and primary hypertension diagnosis in youth. Design, Setting, and Participants: This cross-sectional study included 65â¯452 Delaware Medicaid-insured youths aged 8 to 18 years between January 1, 2014, and December 31, 2019. Residence was geocoded by national area deprivation index (ADI). Exposures: Higher area deprivation. Main Outcomes and Measures: The main outcome was primary hypertension diagnosis based on International Classification of Diseases, Ninth Revision and Tenth Revision codes. Data were analyzed between September 1, 2021, and December 31, 2022. Results: A total of 65â¯452 youths were included in the analysis, including 64â¯307 (98.3%) without a hypertension diagnosis (30â¯491 [47%] female and 33â¯813 [53%] male; mean [SD] age, 12.5 (3.1) years; 12â¯500 [19%] Hispanic, 25â¯473 [40%] non-Hispanic Black, 24â¯565 [38%] non-Hispanic White, and 1769 [3%] other race or ethnicity; 13â¯029 [20%] with obesity; and 31â¯548 [49%] with an ADI ≥50) and 1145 (1.7%) with a diagnosis of primary hypertension (mean [SD] age, 13.3 [2.8] years; 464 [41%] female and 681 [59%] male; 271 [24%] Hispanic, 460 [40%] non-Hispanic Black, 396 [35%] non-Hispanic White, and 18 [2%] of other race or ethnicity; 705 [62%] with obesity; and 614 [54%] with an ADI ≥50). The mean (SD) duration of full Medicaid benefit coverage was 61 (16) months for those with a diagnosis of primary hypertension and 46.0 (24.3) months for those without. By multivariable logistic regression, residence within communities with ADI greater than or equal to 50 was associated with 60% greater odds of a hypertension diagnosis (odds ratio [OR], 1.61; 95% CI 1.04-2.51). Older age (OR per year, 1.16; 95%, CI, 1.14-1.18), an obesity diagnosis (OR, 5.16; 95% CI, 4.54-5.85), and longer duration of full Medicaid benefit coverage (OR, 1.03; 95% CI, 1.03-1.04) were associated with greater odds of primary hypertension diagnosis, whereas female sex was associated with lower odds (OR, 0.68; 95%, 0.61-0.77). Model fit including a Medicaid-by-ADI interaction term was significant for the interaction and revealed slightly greater odds of hypertension diagnosis for youths with ADI less than 50 (OR, 1.03; 95% CI, 1.03-1.04) vs ADI ≥50 (OR, 1.02; 95% CI, 1.02-1.03). Race and ethnicity were not associated with primary hypertension diagnosis. Conclusions and Relevance: In this cross-sectional study, higher childhood neighborhood ADI, obesity, age, sex, and duration of Medicaid benefit coverage were associated with a primary hypertension diagnosis in youth. Screening algorithms and national guidelines may consider the importance of ADI when assessing for the presence and prevalence of primary hypertension in youth.
Assuntos
Hipertensão , Medicaid , Estados Unidos/epidemiologia , Humanos , Masculino , Adolescente , Feminino , Criança , Estudos Transversais , Delaware/epidemiologia , Obesidade , Hipertensão/diagnóstico , Hipertensão/epidemiologia , Hipertensão EssencialRESUMO
PURPOSE: To better define the developmental mechanisms of nonsyndromic cryptorchidism, we measured the expression of hormone receptor and muscle type specific mRNAs in target tissues of boys with and those without nonsyndromic cryptorchidism. MATERIALS AND METHODS: Prospectively collected cremaster muscle and/or hernia sac tissues from boys with congenital (79) or acquired (66) nonsyndromic cryptorchidism and hernia/hydrocele (controls, 84) were analyzed for hormone receptor (RXFP2, AR, ESR1, ESR2) and myosin heavy chain specific (MYH1, MYH2, MYH7) mRNA expression using real-time reverse transcriptase polymerase chain reaction. Log transformed mRNA, phenotype and feeding history data were statistically analyzed using Pearson's correlation, ANOVA and 2-sample t tests. RESULTS: AR mRNA expression was higher in cremaster muscle than in sac tissue, and significantly lower in congenital and acquired nonsyndromic cryptorchidism cases vs controls (p <0.01). Type 1 (slow/cardiac) MYH7 mRNA expression was also significantly reduced in both nonsyndromic cryptorchidism groups (p ≤ 0.002), while a reduction in type 2 (fast) MYH2 expression was more modest and significant only for the congenital cryptorchidism group (p <0.05). Cremasteric MYH7 and AR levels were strongly correlated (r(2) = 0.751, p <0.001). MYH7 and ESR1 mRNA levels were higher and lower, respectively, in boys with nonsyndromic cryptorchidism who were fed soy formula. Expression of other genes was not measurable. CONCLUSIONS: Our data suggest that boys with congenital and acquired nonsyndromic cryptorchidism differentially express AR and slow twitch specific MYH7 mRNA in the cremaster muscle, and that MYH7 expression is correlated with AR levels and soy formula use. These differences in gene expression may reflect aberrant hormonal signaling and/or innervation during development with the potential for secondary functional effects and failed testicular descent.
Assuntos
Criptorquidismo/genética , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , RNA Mensageiro/biossíntese , Receptores Androgênicos/genética , Pré-Escolar , Humanos , Masculino , Músculo Esquelético/química , Estudos Prospectivos , RNA Mensageiro/análise , TestículoRESUMO
Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient-specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC-derived myoblasts (MB) and 1774 CpG sites in differentiating SC-derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo- and hyper-methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.
RESUMO
Cerebral palsy is a set of common, severe, motor disabilities categorized by a static, nondegenerative encephalopathy arising in the developing brain and associated with deficits in movement, posture, and activity. Spastic CP, which is the most common type, involves high muscle tone and is associated with altered muscle function including poor muscle growth and contracture, increased extracellular matrix deposition, microanatomic disruption, musculoskeletal deformities, weakness, and difficult movement control. These muscle-related manifestations of CP are major causes of progressive debilitation and frequently require intensive surgical and therapeutic intervention to control. Current clinical approaches involve sophisticated consideration of biomechanics, radiologic assessments, and movement analyses, but outcomes remain difficult to predict. There is a need for more precise and personalized approaches involving omics technologies, data science, and advanced analytics. An improved understanding of muscle involvement in spastic CP is needed. Unfortunately, the fundamental mechanisms and molecular pathways contributing to altered muscle function in spastic CP are only partially understood. In this review, we outline evidence supporting the emerging hypothesis that epigenetic phenomena play significant roles in musculoskeletal manifestations of CP.
RESUMO
Cord blood (CB) mononuclear cell populations have demonstrated significant promise in biomaterials-based regenerative therapies; however, the contributions of monocyte and macrophage subpopulations towards proper tissue healing and regeneration are not well understood, and the phenotypic responses of macrophage to microenvironmental cues have not been well-studied. In this work, we evaluated the effects of cytokine stimulation and altered substrate stiffness. Macrophage derived from CB CD14+ monocytes adopted distinct inflammatory (M1) and anti-inflammatory (M2a and M2c) phenotypes in response to cytokine stimulation (M1: lipopolysaccharide (LPS) and interferon (IFN-γ); M2a: interleukin (IL)-4 and IL-13; M2c: IL-10) as determined through expression of relevant cell surface markers and growth factors. Cytokine-induced macrophage readily altered their phenotypes upon sequential administration of different cytokine cocktails. The impact of substrate stiffness on macrophage phenotype was evaluated by seeding CB-derived macrophage on 3wt%, 6wt%, and 14wt% poly(ethylene glycol)-based hydrogels, which exhibited swollen shear moduli of 0.1, 3.4, and 10.3 kPa, respectively. Surface marker expression and cytokine production varied depending on modulus, with anti-inflammatory phenotypes increasing with elevated substrate stiffness. Integration of specific hydrogel moduli and cytokine cocktail treatments resulted in the differential regulation of macrophage phenotypic biomarkers. These data suggest that CB-derived macrophages exhibit predictable behaviors that can be directed and finely tuned by combinatorial modulation of substrate physical properties and cytokine profiles.
Assuntos
Sangue Fetal , Macrófagos , Diferenciação Celular , Citocinas , FenótipoRESUMO
OBJECTIVE: Intimal hyperplasia (IH) is the expansion of the vascular intimal region after intervention, which can lead to stenosis and eventual failure of vascular grafts or interventional procedures such as angioplasty or stent placement. Our goals were to investigate the development of IH in a rabbit open surgical model and to evaluate the associated pathophysiological processes involving decorin and the platelet derived growth factor-BB / platelet derived growth factor receptor-ß / mitogen activated protein kinase (PDGF/PDGFR-ß/MAPK) pathway. METHODS: We conducted carotid transection and primary anastomosis on five New Zealand White rabbits to induce IH and examined the associated pathophysiological changes. Tissue was obtained for histological and protein analysis on post-operative day 21 using the contralateral vessel as a control. Intimal medial thickness (IMT) was calculated to measure IH and compared with the unoperated side. Western blot analysis was performed on tissue lysates to determine the expression of decorin core protein, PDGF-BB, PDGFR-ß, and phosphorylated-MAPK (ph-MAPK). Immunofluorescence microscopy was used to assess tissue distribution of matrix metalloproteinase-2 (MMP-2) and phosphorylated-PDGFR-ß (ph-PDGFR-ß). RESULTS: Bilateral carotid arteries were harvested on postoperative day 21. We compared the IMT in operated with unoperated specimens. IMT was significantly elevated in operated arteries vs. unoperated arteries in all 5 animals (148.6 µm +/- 9.09 vs. 103.40 µm +/- 7.08; 135.2 µm +/- 8.30 vs. 92.40 µm +/- 2.35; 203.1 µm +/- 30.23 vs.104.00 µm +/- 4.52; 236.2 µm +/- 27.22 vs. 141.50 µm +/- 9.95; 226.9 µm +/- 11.12 vs. 98.8 µm +/- 3.78). Western blot analysis revealed degradation of decorin protein in the operated tissue, including loss of a 50 kDa band and the appearance of a cleaved fragment at 10 kDa. Decorin and MMP-2 were observed, via immunofluorescence microscopy, in the neointima of the operated vessels. Western blot analysis also revealed increased PDGF-BB, PDGFR-ß, and ph-MAPK levels in operated tissue. Immunofluorescent staining for ph-PDGFR-ß primarily localized to the neointima, indicating increased signaling through PDGF in this region. CONCLUSION: Carotid transection and primary reanastomosis in rabbits induced IH that was associated with MMP-2 activation, degradation of decorin, and activation of the PDGF/PDGFR-ß /MAPK pathway. The findings in this study should lead to further mechanistic evaluation of these pathways to better understand the potential to modify the intimal hyperplastic response to surgery.
RESUMO
Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-ß)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-ß/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.