COL1A1 Gene Research Articles

Combined Alport syndrome, Klinefelter syndrome and Fanconi syndrome in a Chinese boy.

Alport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure. X-linked dominant (XLAS) is the major inheritance form, accounting for almost 80% of the cases, caused by mutations in COL4A5 genes. Klinefelter syndrome (KS) is the most common genetic cause of human male gonadal dysgenesis. AS and KS are both rare disease, there are only three cases of combined AS and KS in the literatures. Fanconi syndrome (FS) caused by AS is also very rare. We report here the first case combined AS, KS and FS in a Chinese boy. We suggest that the severe renal phenotype and FS might be due to the two homozygous COL4A5 variants in our boy, and cases of AS combined KS will be good research objects for X chromosome inactivation.

Transcriptomic analysis of the cerebral hippocampal tissue in spontaneously hypertensive rats exposed to acute hypobaric hypoxia: associations with inflammation and energy metabolism

We evaluated the effect of acute hypobaric hypoxia (AHH) on the hippocampal region of the brain in early-stage spontaneously hypertensive male rats. The rats were classified into a control (ground level; ~ 400 m altitude) group and an AHH experimental group placed in an animal hypobaric chamber at a simulated altitude of 5500 m for 24 h. RNA-Seq analysis of the brains and hippocampi showed that differentially expressed genes (DEGs) were primarily associated with ossification, fibrillar collagen trimer, and platelet-derived growth factor binding. The DEGs were classified into functional categories including general function prediction, translation, ribosomal structure and biogenesis, replication, recombination, and repair. Pathway enrichment analysis revealed that the DEGs were primarily associated with relaxin signaling, PI3K-Akt signaling, and amoebiasis pathways. Protein–protein interaction network analysis indicated that 48 DEGs were involved in both inflammation and energy metabolism. Further, we performed validation experiments to show that nine DEGs were closely associated with inflammation and energy metabolism, of which two (Vegfa and Angpt2) and seven (Acta2, Nfkbia, Col1a1, Edn1, Itga1, Ngfr, and Sgk1) genes showed up and downregulated expression, respectively. Collectively, these results indicated that inflammation and energy metabolism-associated gene expression in the hippocampus was altered in early-stage hypertension upon AHH exposure.

Long-term voluntary exercise inhibited AGE/RAGE and microglial activation and reduced the loss of dendritic spines in the hippocampi of APP/PS1 transgenic mice

Alzheimer’s disease (AD) is closely related to hippocampal synapse loss, which can be alleviated by running exercise. However, further studies are needed to determine whether running exercise reduces synapse loss in the hippocampus in an AD model by regulating microglia. Ten-month-old male wild-type mice and APP/PS1 mice were randomly divided into control and running groups. All mice in the running groups were subjected to voluntary running exercise for four months. After the behavioral tests, immunohistochemistry, stereological methods, immunofluorescence staining, 3D reconstruction, western blotting and RNA-Seq were performed. Running exercise improved the spatial learning and memory abilities of APP/PS1 mice and increased the total number of dendritic spines, the levels of the PSD-95 and Synapsin Ia/b proteins, the colocalization of PSD-95 and neuronal dendrites (MAP-2) and the number of PSD-95-contacting astrocytes (GFAP) in the hippocampi of APP/PS1 mice. Moreover, running exercise reduced the relative expression of CD68 and Iba-1, the number of Iba-1+ microglia and the colocalization of PSD-95 and Iba-1+ microglia in the hippocampi of APP/PS1 mice. The RNA-Seq results showed that some differentially expressed genes (DEGs) related to the complement system (Cd59b, Serping1, Cfh, A2m, and Trem2) were upregulated in the hippocampi of APP/PS1 mice, while running exercise downregulated the C3 gene. At the protein level, running exercise also reduced the expression of advanced glycation end products (AGEs), receptor for advanced glycation end products (RAGE), C1q and C3 in the hippocampus and AGEs and RAGE in hippocampal microglia in APP/PS1 mice. Furthermore, the Col6a3, Scn5a, Cxcl5, Tdg and Clec4n genes were upregulated in the hippocampi of APP/PS1 mice but downregulated after running, and these genes were associated with the C3 and RAGE genes according to protein–protein interaction (PPI) analysis. These findings indicate that long-term voluntary exercise might protect hippocampal synapses and affect the function and activation of microglia, the AGE/RAGE signaling pathway in microglia and the C1q/C3 complement system in the hippocampus in APP/PS1 mice, and these effects may be related to the Col6a3, Scn5a, Cxcl5, Tdg and Clec4n genes. The current results provide an important basis for identifying targets for the prevention and treatment of AD.

Collagen XII mediated cellular and extracellular mechanisms in development, regeneration, and disease.

Collagen XII, a fibril-associated collagen with interrupted triple helices (FACIT), influences fibrillogenesis in numerous tissues. In addition to this extracellular function, collagen XII also directly regulates cellular function. Collagen XII is widely expressed in connective tissues, particularly tendons, ligaments, and the periodontium and periosteum, where it is enriched in the pericellular regions. Mutations in the collagen XII gene cause myopathic Ehlers-Danlos syndrome (mEDS), an early-onset disease characterized by overlapping connective tissue abnormalities and muscle weakness. Patients with mEDS exhibit delayed motor development, muscle weakness, joint laxity, hypermobility, joint contractures, and abnormal wound healing. A mEDS mouse model was generated by deletion of the Col12a1 gene, resulting in skeletal and muscle abnormalities with disorganized tissue structures and altered mechanical properties. Extracellularly, collagen XII interacts with collagen I fibrils and regulates collagen fibril spacing and assembly during fibrillogenesis. Evidence for the binding of collagen XII to other EDS-related molecules (e.g., decorin and tenascin X) suggests that disruption of ECM molecular interactions is one of the causes of connective tissue pathology in mEDS. Collagen XII also has been shown to influence cell behavior, such as cell shape and cell-cell communication, by providing physical connection between adjacent cells during tissue development and regeneration. The focus of this review is on the functions of collagen XII in development, regeneration, and disease.

Molecular dynamics and minigene assay of new splicing variant c.4298-20T>A of COL4A5 gene that cause Alport syndrome.

Introduction: Alport syndrome (AS; OMIM#308940) is a progressive hereditary kidney disease characterized by hearing loss and ocular abnormalities. According to the mode of inheritance, AS has three subtypes: X-linked (XL; OMIM#301050), autosomal recessive (AR; OMIM#203780), and autosomal dominant (AD; OMIM#104200). XLAS is caused by a pathogenic variant in COL4A5 (OMIM*303630) gene encoding type IV collagen (Col-IV) α5 chain, while ADAS and ARAS are consequences of a variant in COL4A3 (OMIM*120070) and COL4A4 (OMIM*120131) genes that encode Col-IV α3 and α4 chains, respectively. Usually, diagnosis of AS requires hereditary or pathological examinations. Splicing variants are hard to be determined as pathogenic or non-pathogenic based on the results of gene sequencing. Methods: This study focused on a splicing variant in COL4A5 gene, termed NM_000495.5: c.4298-20T>A, and to analyzed its authenticity and damaged α5 chain. In vitro minigene splicing assay was applied to investigate the effect of splicing variant, c.4298-20T>A, on COL4A5 mRNA synthesis. Molecular dynamics method was used to predict the capability of the responsible α5(IV) to form a triple helix. Results: The intron 46 of COL4A5 mRNA retained 18 bp, resulting in insertion of six amino acids behind the amino acid at position 1,433 of α5(IV). The predicted protein effect of this variant: p. (Pro1432_Gly1433insAspTyrPheValGluIle). As a consequence, the stability of α5(IV) secondary structure was impaired, probably leading to the unusual configuration of α345(IV). Discussion: Normally, splicing variant in COL4A5 gene can lead to phenotypes of XLAS, and the effect is associated with the extent of splicing. The patient reported here carried a c.4298-20T>A splicing variant in COL4A5 gene, and AS was highly suspected based on the pathology results. However, the patient did not manifest any ocular or ear abnormalities. We therefore present the c.4298-20T>A splicing variant in COL4A5 gene as likely-pathogenic splicing variant that leads to XLAS with mild phenotypes.

Comparing COL7A1 gene expression in fibroblast cells of dystrophic epidermolysis bullosa patients with clinical responses to autologous fibroblasts transplantation

Objectives: This clinical research aimed to establish autologous fibroblasts transplantation as a possible treatment for patients with DEJ. The COL7A1 gene expression was also evaluated.
 Methods: Six patients (3M and 3F), 4 with no recurrent wounds and 2 with recurrent wounds after surgery, and 15 healthy subjects were included in the study as controls. Quantitative real-time polymerase chain reaction (real-time PCR) analysis of the COL7A1 gene was performed using an oligonucleotide primer pair designed to amplify across the exon/exon junction.
 Results: The COL7A1 expression level was down-regulated at exons 26-27, 47-48, 96-97, and 116-117 in all patients′ fibroblasts compared with the healthy controls. However, the expression of the COL7A1 gene in the fibroblasts of the patients with a positive response to the treatment was not significantly changed compared with the patients with the poor response. (ClinicalTrials.gov NCT01908088)
 Conclusion: In this study the mRNA expression levels of COL7A1 were significantly less in the patients when compared with healthy controls. However the COL7A1 expression after autologous fibroblasts transplantation was not different between the two groups of patients, and further examination is needed to elucidate the mechanism of the treatment.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells

ABSTRACT Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-β1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism.

Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism.

Trio whole-exome sequencing reveals a novel de novo mutation in COL2A1 gene in an Iranian patient with hypochondroplasia

Hypochondroplasia is a hereditary form of skeletal dysplasia characterized by very short stature, chest with short ribs, and disproportionately legs and arms. The COL2A1 gene encodes the alpha-1 chain of type II collagen and is the major structural part of the extracellular matrix. A variety of alterations in this gene have been shown to be responsible as the cause of skeletal dysplasia and cartilage defect. In this study, we performed a trio-analysis of whole-exome sequencing (WES) and in silico investigation for an Iranian patient with hypochondroplasia and her parents. The DNA samples were extracted from an 8 years old female patient and her parents. WES was conducted for these samples and the results were eventually confirmed and segregated via Sanger sequencing. Our findings demonstrated a novel de novo heterozygous missense variant in COL2A1 gene, chr12:48373338:C > T, NM_033150, c.G2482A, (P.G828R). By Sanger sequencing, this disease-causing variant was confirmed in the patient, while parents' Sanger results showed the presence of wild-type alleles of the gene as was introduced via WES. Additionally, computational databases predicted damaging effects for this mutation. Results of this study expand the variant frequency of COL2A1 gene and also exhibit foundational clues for genetic counselling and future hypochondroplasia diagnosis and therapy.

Pontine autosomal dominant microangiopathy with leukoencephalopathy: Col4A1 gene variants in the original family and sporadic stroke

Background(1) Description of clinical and cranial MRI features in the original Pontine Autosomal Dominant Microangiopathy with Leukoencephalopathy (PADMAL) family and correlation with the segregation analysis of the causative collagen 4A1 gene (COL4A1) variant. (2) Sequence analysis of the COL4A1 miRNA-binding site containing the causative variant in two independent cross-sectional samples of sporadic stroke patients.Patients and methodsSanger sequencing of the COL4A1 miRNA-binding site in the PADMAL family and 874 sporadic stroke patients.ResultsPADMAL shows adult-onset usually between 30 and 50 years of age with initial brainstem-related symptoms most commonly dysarthria, with progression to dementia and tetraparesis. Radiologically pontine lacunes are followed by supratentorial white matter involvement. Radiological onset may precede clinical symptoms. We found no variants in the COL4A1 miRNA-binding site of sporadic stroke patients.ConclusionOur results allow an early diagnosis of PADMAL based on cranial MRI, clinical signs, and confirmatory sequencing of the COL4A1 miRNA-29-binding site. COL4A1 miRNA-29-binding site variants do not contribute to a sizeable proportion of sporadic stroke.

Blood Inflammatory-like and Lung Resident-like Eosinophils Affect Migration of Airway Smooth Muscle Cells and Their ECM-Related Proliferation in Asthma

Airway remodeling is a hallmark feature of asthma, and one of its key structural changes is increased airway smooth muscle (ASM) mass and disturbed extracellular matrix (ECM) homeostasis. Eosinophil functions in asthma are broadly defined; however, we lack knowledge about eosinophil subtypes' interaction with lung structural cells and their effect on the airway's local microenvironment. Therefore, we investigated the effect of blood inflammatory-like eosinophils (iEOS-like) and lung resident-like eosinophils (rEOS-like) on ASM cells via impact on their migration and ECM-related proliferation in asthma. A total of 17 non-severe steroid-free allergic asthma (AA), 15 severe eosinophilic asthma (SEA) patients, and 12 healthy control subjects (HS) were involved in this study. Peripheral blood eosinophils were enriched using Ficoll gradient centrifugation and magnetic separation, subtyped by using magnetic separation against CD62L. ASM cell proliferation was assessed by AlamarBlue assay, migration by wound healing assay, and gene expression by qRT-PCR analysis. We found that blood iEOS-like and rEOS-like cells from AA and SEA patients' upregulated genes expression of contractile apparatus proteins, COL1A1, FN, TGF-β1 in ASM cells (p < 0.05), and SEA eosinophil subtypes demonstrated the highest effect on sm-MHC, SM22, and COL1A1 gene expression. Moreover, AA and SEA patients' blood eosinophil subtypes promoted migration of ASM cells and their ECM-related proliferation, compared with HS (p < 0.05) with the higher effect of rEOS-like cells. In conclusion, blood eosinophil subtypes may contribute to airway remodeling by upregulating contractile apparatus and ECM component production in ASM cells, further promoting their migration and ECM-related proliferation, with a stronger effect of rEOS-like cells and in SEA.

Exploration of Novel Genetic Evidence and Clinical Significance Into Hemifacial Microsomia Pathogenesis

The authors browsed through past genetic findings in hemifacial microsomia along with our previously identified mutations in ITGB4 and PDE4DIP from whole genome sequencing of hemifacial microsomia patients. Wondering whether these genes influence mandibular bone modeling by regulation on osteogenesis, the authors approached mechanisms of hemifacial microsomia through this investigation into gene knockdown effects in vitro. MC3T3E1 cells were divided into 5 groups: the negative control group without osteogenesis induction or siRNA, the positive control group with only osteogenesis induction, and 3 gene silenced groups with both osteogenesis induction and siRNA. Validation of transfection was through fluorescence microscopy and quantitative real-time Polymerase chain reaction on knockdown efficiency. Changes in expression levels of the 3 genes during osteogenesis and impact of Itgb4 and Pde4dip knockdown on osteogenesis were examined by quantitative real-time Polymerase chain reaction, alkaline phosphatase, and alizarin red staining. Elevation of osteogenic genes Alpl, Col1a1, Bglap, Spp1, and Runx2 verified successful osteogenesis. Both genes were upregulated under osteogenic induction, while they had different trends over time. Intracellular fluorophores under microscope validated successful transfection and si-m-Itgb4_003, si-m-Pde4dip_002 had satisfactory knockdown effects. During osteogenesis, Pde4dip knockdown enhanced Spp1 expression (1.95±0.13 folds, P =0.045). The authors speculated that these genes may have different involvements in osteogenesis. Stimulated expression of Spp1 by Pde4dip knockdown may suggest that Pde4dip inhibits osteogenesis.

Paraspinal muscle gene expression across different aetiologies in individuals undergoing surgery for lumbar spine pathology

PurposeThe purpose of this study was to understand potential baseline transcriptional expression differences in paraspinal skeletal muscle from patients with different underlying lumbar pathologies by comparing multifidus gene expression profiles across individuals with either disc herniation, facet arthropathy, or degenerative spondylolisthesis.MethodsMultifidus biopsies were obtained from patients (n = 44) undergoing lumbar surgery for either disc herniation, facet arthropathy, or degenerative spondylolisthesis. Diagnostic categories were based on magnetic resonance images, radiology reports, and intraoperative reports. Gene expression for 42 genes was analysed using qPCR. A one-way analysis of variance was performed for each gene to determine differences in expression across diagnostic groups. Corrections for multiple comparisons across genes (Benjamini–Hochberg) and for between-group post hoc comparisons (Sidak) were applied.ResultsAdipogenic gene (ADIPOQ) expression was higher in the disc herniation group when compared to the facet arthropathy group (p = 0.032). Adipogenic gene (PPARD) expression was higher in the degenerative spondylolisthesis group when compared to the disc herniation group (p = 0.013), although absolute gene expression levels for all groups was low. Fibrogenic gene (COL3A1) had significantly higher expression in the disc herniation group and facet arthropathy group when compared to the degenerative spondylolisthesis group (p < 0.001 and p = 0.038, respectively). When adjusted for multiple comparisons, only COL3A1 remained significant (p = 0.012).ConclusionIndividuals with disc herniation and facet arthropathy demonstrate higher COL3A1 gene expression compared to those with degenerative spondylolisthesis. Future research is required to further understand the biological relevance of these transcriptional differences.

Investigation of Genetic Mutations in the Exon-intron Regions of the COL7A1 Gene in five Family Case Reports with Epidermolysis Bullosa in Khuzestan Province

Background: Dystrophic Epidermolysis bullosa (DEB) is one of the EB types caused by a mutation in the COL7A1 gene, which encodes collagen type 7 and is the main component of anchoring fibrils. Objectives: This study aimed to investigate COL7A1 gene mutations in five families of patients. Khuzestan is suffering from EB. Methods: After extracting genomic DNA from peripheral blood samples of probands, COL7A1 gene screening was performed using polymerase chain reaction and direct sequencing of exons 36-37 and their adjacent introns with the Sanger sequencing method. Results: The identified mutations included a new homozygous deletion mutation in exon 36 (c.2669delC) that resulted in a change in the open reading frame. Other obtained variants are the common mutations c.4233delC and c.4233delT in exon 37, which were identified as proline substitution mutations in one mutant allele in the homozygous form and three allele mutants in the heterozygous form. Conclusions: In this study, presenting a new mutation in the COL7A1 gene is the focus of the molecular heterogeneity of DEB. In addition, the efficient identification of COL7A1 gene mutations provides more information for the accurate diagnosis and prediction of the disease, genetic counseling, and prenatal diagnosis.

Abstract TMP23: Fetal Intraparenchymal Hemorrhage In Col4a1/2-related Disorders

Introduction: COL4A1/A2 mutations affecting the alpha 1 and 2 chains of type IV collagen are increasingly recognized as a potential cause of intracranial hemorrhage across the lifespan. We aim to describe fetal MRI findings and clinical outcomes in seven cases of fetal intraparenchymal hemorrhage (IPH) associated with COL4A1/A2 mutations. Methods: Subjects with COL4A1/A2 mutations were identified from a retrospective single-center cohort study of fetal IPH identified on fetal brain MRI between 1998-2021. Imaging findings were reviewed by two pediatric neuroradiologists, and clinical data were collected from electronic medical records. Results: Among the seven subjects with fetal IPH and COL4A1/A2 mutations, fetal MRI was performed at median gestational age (GA) 29 weeks (range 22-36 weeks) and demonstrated supratentorial IPH in 7/7 with variable size of hemorrhage, including 4/7 with multifocal hemorrhage (Figure 1). Four fetuses were liveborn at median GA 39 weeks (range 33-42 weeks); three pregnancies were terminated. Postnatal imaging revealed recurrent IPH in 1/4 cases, but no cases had recurrent IPH after the neonatal period. None of the seven cases had known family history of COL4A1/A2 or IPH; genetic testing revealed 5/7 with COL4A1 mutation, 1/7 with COL4A2 mutation, and 1/7 with 13q34 deletion with associated loss of COL4A1 and COL4A2 genes. Clinical follow-up data was available for the four liveborn cases through ≥12 months; three had mild hemiplegic cerebral palsy and one had severe spastic quadriplegia. None required CSF diversion, none had epilepsy, and one had visual impairment. Conclusions: Our series suggests that COL4A1/A2 mutations can present as fetal IPH with a wide range of phenotypes ranging from unifocal to multifocal IPH without additional systemic involvement, and that prenatal imaging severity may be associated with early clinical outcomes. COL4A1/A2 testing should be considered in all cases of prenatal IPH, regardless of severity.

An animal model study of osteochondral defect repair by human adipose stem cells and pomegranate fruit hydroalchoholic extract.

Articular cartilage damages do not repair spontaneously. Tissue engineering is a promising approach to repair cartilage damage. Transforming growth factor-beta (TGF-β) members are the known induction factors in chondrogenic differentiation. However, hypertrophy of the chondrocytes resulting from mesenchymal stem cells (MSCs) induction by TGF-β is inevitable. Pomegranate fruit contains many ingredients which are useful in ensuring the health of organs. This study was designed to investigate the Pomegranate Fruit hydroalchoholic Extract (PFE) capability in human adipose derived stem cells (hASCs) differentiation into the chondrocytes on fibrin scaffold. Pomegranate fruit hydroalchoholic extract (PFE) was prepared. hASCs were isolated, expanded, labeled, and seeded on the fibrin scaffold. The constructs were divided into three groups including TGF-β3, PFE, and control. The constructs were induced for 14 days, then, the MTT assay, Real-Time Polymerase Chain Reaction (PCR), and histochemistry assessments were run, and finally, the constructs were transplanted into the knee defect of rats. The gross and histological assessments of the transplants were done after 8 weeks. The viability rate, COL2A1, Aggrecan (ACAN) and COL10A1 genes expression levels, and histological criterion of the PFE samples were significantly higher than that of the control. The macroscopic grades and histological results of the PFE samples were close to that of the TGF-β3. The number of positive cells for COLІI protein were higher significantly in the PFE group than the control. PFE was effective in the chondrogenic induction of hASCs. Further studies are needed to find out the events of the chondrogenic induction using PFE.

Overexpression of miR-148a-3p inhibits extracellular matrix degradation and alleviates IL-1β-induced intervertebral disc degeneration.

Recently, studies on microRNAs (miRNAs) and their targets and related genes have provided new therapeutic opportunities for controlling intervertebral disc degeneration (IDD). We aimed to investigate the effects of miR-148a-3p overexpression on IDD progression. This study used microRNA microarrays to analyze key regulators of IDD. Q-PCR was used to verify the IL-1β-induced down-regulation of miR-148a-3p expression both in nucleus pulposus (NP) tissues of IDD patients and in degenerated NP cells (NPCs) of rats. Rat NPC micromass cultures and ex vivo intervertebral disc (IVD) culture models were established, and histological staining was performed to verify the effect of miR-148a-3p on the general morphology and proteoglycan and collagen contents of IVDs. In addition, q-PCR and western blotting analyses were performed to examine the expression of ECM molecules and matrix-degrading enzymes. A luciferase reporter assay was used to confirm the target genes of miR-148a-3p. Our data revealed that miR-148a-3p was down-regulated in IDD. Overexpression of miR-148a-3p had no effect on ACAN or COL2A1 gene expression but decreased MMP3, MMP13, and ADAMTS5 gene expression. The matrix deposited by miR-148a-3p-overexpressing rat NPCs contained high levels of proteoglycans and collagen. The ex vivo experiments verified that agomiR-148a-3p alleviated the NPC matrix degradation induced by IL-1β. A luciferase reporter assay confirmed that miR-148a-3p directly targeted ADAMTS5 and MMP13. We proved that miR-148a-3p can attenuate ECM loss and protect NP function by inhibiting matrix-degrading enzymes.