Collagen Gene Research Articles

Differential network analysis reveals the key role of the ECM-receptor pathway in α-particle-induced malignant transformation

Space particle radiation is a major environmental factor in spaceflight, and it is known to cause body damage and even trigger cancer, but with unknown molecular etiologies. To examine these causes, we developed a systems biology approach by focusing on the co-expression network analysis of transcriptomics profiles obtained from single high-dose (SE) and multiple low-dose (ME) α-particle radiation exposures of BEAS-2B human bronchial epithelial cells. First, the differential network and pathway analysis based on the global network and the core modules showed that genes in the ME group had higher enrichment for the extracellular matrix (ECM)-receptor interaction pathway. Then, collagen gene COL1A1 was screened as an important gene in the ME group assessed by network parameters and an expression study of lung adenocarcinoma samples. COL1A1 was found to promote the emergence of the neoplastic characteristics of BEAS-2B cells by both in vitro experimental analyses and in vivo immunohistochemical staining. These findings suggested that the degree of malignant transformation of cells in the ME was greater than that of the SE, which may be caused by the dysregulation of the ECM-receptor pathway.

LncRNA CDKN2B-AS1 regulates collagen expression

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10− 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10− 25). The inflammatory “TNFA signaling via NFKB” gene set was downregulated the most (Padj=1 × 10− 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10− 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10− 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.

#2438 ADPKD and collagen genes (COL4A3, COL4A4, COL4A5)

Abstract Background and Aims Familial hematuria diseases are a heterogeneous group of monogenic conditions caused by mutations in one of the collagen IV genes: COL4A3 (2q36.3), COL4A4 (2q36.3), and COL4A5 (Xq22.3) that are expressed in the glomerular basement membranes (GBM) and are responsible for the most frequent forms of microscopic hematuria (MH), Alport syndrome, and thin basement membrane nephropathy (TBMN). Recent data suggest that about 1% (1 in 106 individuals) of the world population may have heterozygous predicted pathogenic COL4A3 or COL4A4 variants, a frequency that leads to the occasional superimposition of TBMN with other glomerulopathies. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major genetic disorder affecting up to 12.5 million individuals worldwide and it is the fourth most common global cause of renal replacement therapy. Two are the principal causative genes: PKD1(16p13.3) and PKD2 (4q22.1). The aim of the study was to describe a cohort of clinically ADPKD patients studying collagen IV genes COL4A3, COL4A4, and COL4A5. Method We performed NGS with Sophia Genetic “Nephropathies Solution” Panel on clinically ADPKD patients (pts). This panel includes 44 genes (target region 105.8 kb) involved in different types of nephropathies. We considered variants that are categorized as Variant of Uncertain Significance (Vus), likely pathogenic, or pathogenic (Class 3,4, or 5 of American College of Medical Genetics (ACMG)) in COL4A3, COL4A4, and COL4A5 genes. Results We analyzed 250 consecutive clinically ADPKD pts (consecutive in the outpatient clinic). We found 13 (6.3%) clinically potentially interesting (Class 3,4 or 5 of ACMG) variants in collagen genes. We found 2 pts that were negative for PKD1, PKD2, PKHD1 analysis: 1 pt carrier COL4A4 NM_000092.5: c.2908C&amp;gt;T (p.Gln970Ter), Pathogenic and c.2756A&amp;gt;G (p.Glu919Gly) Vus; 1 male pt carrier COL4A3 NM_000091.5: c.609+3_609+6del, Vus and COL4A5 NM_000495:c.169G&amp;gt;A p.(Gly57Arg), likely pathogenic. We also found 4 pts with 4 Vus variants in collagens genes combined with at least 1 pathogenic or likely pathogenic variant in PKD1 or PKD2 genes. We found 3 pts with likely pathogenic variants in COL4A4 or COL4A3 genes combined with Vus or likely benign variants in PKD1 or PKD2 genes and 1 patient with 1 likely pathogenic variant in COL4A3 and 1 likely pathogenic variant in PKD1. Conclusion Variants in collagen genes are very frequent and the associated phenotype is varied, sometimes very mild such as microhematuria. Furthermore, penetrance has to be considered. The study outlines the importance of considering also collagen genes, even if we have a clear clinical manifestation of ADPKD. An additional variant in a kidney already compromised can have an impact on the phenotype. Analysis of genes involved in these 2 pathologies could lead to a better understanding of the phenotype-genotype correlation at least in patients with both conditions.

Identification of collagen subtypes of gastric cancer for distinguishing patient prognosis and therapeutic response.

Gastric cancer is a highly heterogeneous disease, presenting a major obstacle to personalized treatment. Effective markers of the immune checkpoint blockade response are needed for precise patient classification. We, therefore, divided patients with gastric cancer according to collagen gene expression to indicate their prognosis and treatment response. We collected data for 1250 patients with gastric cancer from four cohorts. For the TCGA-STAD cohort, we used consensus clustering to stratify patients based on expression levels of 44 collagen genes and compared the prognosis and clinical characteristics between collagen subtypes. We then identified distinct transcriptomic and genetic alteration signatures for the subtypes. We analyzed the associations of collagen subtypes with the responses to chemotherapy, immunotherapy, and targeted therapy. We also established a platform-independent collagen-subtype predictor. We verified the findings in three validation cohorts (GSE84433, GSE62254, and GSE15459) and compared the collagen subtyping method with other molecular subtyping methods. We identified two subtypes of gastric adenocarcinoma: a high-expression collagen subtype (CS-H) and a low-expression collagen subtype (CS-L). Collagen subtype was an independent prognostic factor, with better overall survival in the CS-L subgroup. The inflammatory response, angiogenesis, and phosphoinositide 3-kinase (PI3K)/Akt pathways were transcriptionally active in the CS-H subtype, while DNA repair activity was significantly greater in the CS-L subtype. PIK3CA was frequently amplified in the CS-H subtype, while PIK3C2A, PIK3C2G, and PIK3R1 were frequently deleted in the CS-L subtype. CS-H subtype tumors were more sensitive to fluorouracil, while CS-L subtype tumors were more sensitive to immune checkpoint blockade. CS-L subtype was predicted to be more sensitive to HER2-targeted drugs, and CS-H subtype was predicted to be more sensitive to vascular endothelial growth factor and PI3K pathway-targeting drugs. Collagen subtyping also has the potential to be combined with existing molecular subtyping methods for better patient classification. We classified gastric cancers into two subtypes based on collagen gene expression and validated these subtypes in three validation cohorts. The collagen subgroups differed in terms of prognosis, clinical characteristics, transcriptome, and genetic alterations. The subtypes were closely related to patient responses to chemotherapy, immunotherapy, and targeted therapy.

Serum miR‑29 is increased in mice with early liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is a fatty liver disease that is not caused by alcohol consumption and is characterized by fatty degeneration, inflammation and hepatocellular damage. Therefore, predicting future fibrosis is critical in the early stages of NASH to prevent disease progression. The present study examined histological changes in the liver as well as microRNA (miR/miRNA) expression changes in the liver and serum of NASH mice model to identify potential biomarker candidates that could predict early fibrosis. This study used 6-week-old C57BL/6NJcl male mice and fed the control with a standard solid diet (CE-2) for breeding and propagation and NASH groups with a high-fat diet [choline-deficient high-fat and 0.1% (w/v) methionine supplemented diet], respectively. Agilent Technologies miRNA microarray was used to investigate microRNA expression in the liver and serum. Hematoxylin and eosin staining of the livers of the NASH group mice during the second week of feeding revealed fatty degeneration, balloon-like degeneration and inflammatory cell infiltration, confirming that the mice were in a state of NASH. The livers of the NASH group mice at 6 weeks of feeding showed fibrosis. Microarray analysis revealed that miRNAs were upregulated and 47 miRNAs were downregulated in the liver of the NASH group. Pathway analysis using OmicsNet predicted miR-29 to target collagen genes. Furthermore, miR-29 was downregulated in the livers of NASH-induced mice but upregulated in serum. These findings suggested that lower miR-29 expression in NASH-induced liver would increase collagen expression and fibrosis. Early liver fibrosis suggests that miR-29 leaks from the liver into the bloodstream, and elevated serum miR-29 levels may be a predictive biomarker for early liver fibrosis.

Significant improvement of cardiac outflow tract septation defects in a DiGeorge syndrome model after minoxidil treatment

The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation.We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1−/− mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant.We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

The fibroid phenotype of biological naïve patients with rheumatoid arthritis are less likely to respond to anti-IL-6R treatment

Type III collagen gene expression is upregulated in the synovium of patients with rheumatoid arthritis (RA) presenting the fibroid phenotype. The soluble type III collagen formation biomarker, PRO-C3, is known to measure fibrogenesis in fibrotic diseases. In this exploratory study, we aimed to investigate the association between fibrogenesis (PRO-C3) and the disease- and treatment response in patients with RA. We measured PRO-C3 in subsets of two clinical trials assessing the effect of the anti-interleukin-6 (IL-6) receptor treatment tocilizumab (TCZ) as monotherapy or polytherapy with methotrexate. PRO-C3 levels had weak or very weak correlations with the clinical parameters (Spearman’s). However, when the patients were divided into Disease Activity Score-28 groups characterized by the erythrocyte sedimentation rate (DAS28-ESR), there was a statistical difference between the PRO-C3 levels of the different groups (p < 0.05). To determine the response in relation to PRO-C3, a cut-off based on PRO-C3 levels and patients in remission (DAS28-ESR ≤ 2.6) was identified. This showed that a reduction in PRO-C3 after treatment initiation was associated with decreased DAS28-ESR and a higher response rate in patients with low PRO-C3 levels than in those with high PRO-C3 levels. This indicates that a fibrotic component affects the responsiveness of patients.

ZNF469 is a profibrotic regulator of extracellular matrix in hepatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) into proliferative myofibroblasts drives extracellular cellular matrix (ECM) accumulation and liver fibrosis; nevertheless, the transcriptional network that promotes such a process is not completely understood. ZNF469 is a putative C2H2 zinc finger protein that may bind to specific genome sequences. It is found to be upregulated upon HSC activation; however, the molecular function of ZNF469 is completely unknown. Here, we show that knockdown of ZNF469 in primary human HSCs impaired proliferation, migration, and collagen production. Conversely, overexpression of ZNF469 in HSCs yielded the opposite results. Transforming growth factor-β 1promoted expression of ZNF469 in a Smad3-dependent manner, where the binding of Smad3 was confirmed at the ZNF469 promoter.RNA sequencingdata of ZNF469-knockdown HSCs revealed the ECM-receptor interaction, which provides structural and signaling support to cells, was the most affected pathway, and significant downregulation of various collagen and proteoglycan genes was observed. To investigate the function of ZNF469, we cloned a full-length open reading frame of ZNF469 with an epitope tag and identified a nuclear localization of the protein. Luciferase reporter and chromatin immunoprecipitationassays revealed the presence of ZNF469 at the promoter of ECM genes, supporting its function as a transcription factor. Analysis of human fibrotic and cirrhotic tissues showed increased expression of ZNF469 and a positive correlation between expression levels of ZNF469 and ECM genes. Moreover, this observation was similar in other fibrotic organs, including the heart, lung, and skin, suggesting that myofibroblasts from various origins generally require ZNF469 to promote ECM production. Together, this study is the first to reveal the role of ZNF469 as a profibrotic factor in HSCs and suggests ZNF469 as a novel target for antifibrotic therapy.

Abstract PO4-26-03: Collagen deposition in the breast cancer microenvironment correlates with ER-positive, Histological Grade I and low proliferation tumors

Abstract Breast cancer is a highly heterogeneous neoplasm both at the molecular and histological levels. Breast cancer cells present extremely high levels of genetic heterogeneity concerning mutational burden, copy number variations (CNVs) and epigenetic signatures. While this level of heterogeneity is well established, other layers of heterogeneity, such as tumor microenvironment (TME) heterogeneity, remains to be elucidated. TME in breast cancer is an intricate network encompassing tumor cells, “normal” stroma cells such as fibroblasts, lymphocytes, and a plethora of extracellular matrix (ECM) proteins produced either by cancer cells or stromal cells. These three interacting modules (cancer cells, stromal cells and the ECM) can affect each other in a reciprocal way with profound effects on patient prognosis and treatment outcome. Major protein components of the TME are ECM-related collagens, that are produced by breast cancer cells or/and cancer associated fibroblasts. The ECM collagen in breast cancer, that is mainly composed of collagen types I, III and V, has multiple roles modulating tumor growth, cancer cell invasiveness and metastasis. Here by Masson Trichrome and Immunohistochemistry/Aniline Blue co-stains for collagen fibers and proliferation index (Ki-67) in breast cancer FFPE tissue samples, we identified a statistically significant correlation between collagen deposition and Estrogen Receptor (ER)) status (p&amp;lt; 0.05, proliferation index (p&amp;lt; 0.01) and histological Grade (p=0.01). Further, in order to expand and support our findings, we analyzed the METABRIC study through the cBioPortal hub in relevance to collagen genes and multiple clinicopathological parameters. We were able to identify a coregulated subset of collagen genes that presented a positive correlation with ER-positive (p&amp;lt; 10−10, q&amp;lt; 10−10), low proliferating (p&amp;lt; 10−10, q&amp;lt; 10−10), Grade I (p=2.35e−9, q&amp;lt; 1.07e−8) group of breast cancer cases, supporting our findings. In addition, analyses of collagen-high patients from the METABRIC study indicated that most cases presented with PIK3CA, CBFB and MAP3K1 mutations, while through the g.Profiler software we identified collagen-high relevant gene signatures that encompassed gene ontology terms related to tissue development, extracellular matrix organization and cell migration. Collectively, our preliminary results indicate that ECM collagen deposition in breast cancer is characteristic of low-grade tumors with a better differentiation and possibly with a favorable prognosis. Citation Format: Angelos Koutras, Chaido Sirinian, Olga Bogri, Martha Nifora, Maria Theakou, Soren Degn, Ioannis Mouratidis, Dimitrios Chaniotis, Stavros Peroukidis, Ilias Georgakopoulos-Soares, Anastasios Papanastasiou. Collagen deposition in the breast cancer microenvironment correlates with ER-positive, Histological Grade I and low proliferation tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-03.

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability-Brief Report.

Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.

Characterizing the association of systemic estradiol levels and transcriptional markers of skeletal muscle regeneration in healthy young females

Recent research indicates estradiol's (E2) role in modulating skeletal muscle regeneration. E2 has been observed to enhance myogenic capacity by upregulating myogenic factors and downregulating proteolytic factors[1,2]. The extracellular matrix (ECM) of skeletal muscle also influences regeneration due to its interaction with muscle progenitor cells [5]. Cell and animal studies observed E2's ability to impact on collagen (COL) 1, 3, and 4 production [3,4]. However, limited data from physiologically relevant human models exploring myogenic markers and ECM throughout the menstrual cycle are available. We hypothesize that higher systemic E2 levels will correspond to an increased regenerative gene expression signature. Understanding this relationship is crucial for assessing how E2 levels affect muscle healing in eumenorrheic women and E2’s potential as a therapeutic molecule for enhancing muscle repair. The study included 19 pre-menopausal women aged 18-39 undergoing hip surgery at UT Southwestern (UTSW) Medical Center. This study was approved by the UTSW IRB and conducted in accordance with the Declaration of Helsinki. Blood and muscle biopsies were collected for hormone and gene analysis. Only subjects in the follicular phase (n=12) were included to minimize progesterone's influence. Investigated genes included myogenic factors (MYOD1, MYF5, MYF6, MYOG), proteolytic factors (FOXO3, MuRF1), and COL genes (COL1a1, COL-3a1, COL-4a1, COL-6a1). E2 levels ranged from 26-356 pg/mL, with negligible progesterone levels, consistent with the follicular phase. While no significant relationships were found between myogenic and proteolytic genes, MYF6 exhibited a positive relationship with E2 (p=0.07), while MuRF1 showed a negative one (p=0.09). COL 1, 3, and 6 gene expressions had no significant relationship with E2, but COL 4 exhibited a statistically significant positive relationship (p=0.04) with E2 concentration. Though no statistically significant relationships emerged between myogenic or proteolytic gene expression and E2 levels, there were notable trends toward significance for MYF6 and MuRF1. MYF6, recognized for its role in myogenic differentiation, is implicated in maintaining the satellite cell pool by promoting myokine secretion[6]. In contrast, MuRF1 is a critical component in muscle atrophy[7]. COL4 expression was significantly associated with E2 levels (p=0.4). It is hypothesized that COL4 enhances IGF1-activated myoblast migration, differentiation, and fusion [5]. These findings suggest a potential link between elevated E2 levels and an enhanced regenerative capacity. These findings indicate that higher E2 conditions may create a favorable environment for muscle recovery. Future research should increase sample size and examine protein levels of these factors.

Abstract 133: Trem2 Agonist Promotes Atherosclerotic Plaque Stability

Atherosclerosis is an inflammatory disease of the arteries that is a major cause of cardiovascular disease (CVD). Atherosclerotic plaque progression is driven by the deposition of low-density lipoprotein (LDL) within the intima, which is internalized by macrophages, forming lipid-engorged foamy cells. Importantly, the accumulation of excess LDL within foamy macrophages is cytotoxic, driving apoptosis, which ultimately promotes necrotic core formation and increased potential for plaque rupture. Recently, our lab identified the lipid receptor Trem2 to be highly expressed by foamy macrophages and regulate cell survival, proliferation, and cholesterol efflux. Thus, we tested whether agonizing Trem2 could drive macrophage survival to promote plaque stability. Using a Trem2 agonist antibody (AL002a, Alector Inc.), we treated Ldlr -/- mice that had preexisting atherosclerotic plaque with either isotype control or AL002a antibody for 8 weeks. AL002a treated mice showed an increase in plaque size in the aortic arch and aortic sinus. Plaque analysis determined that this was primarily due to an expansion of macrophage cellularity, through promotion of cell survival and proliferation. Furthermore, we found that plaques from AL002a treated mice had decreased necrotic core formation, increased fibrous cap size, and increased collagen deposition, consistent with improved stability. Single cell RNA-sequencing of atherosclerotic aortae from isotype and AL002a treated mice found that Trem2-agonism dramatically reshaped transcriptomes of both foamy macrophages and smooth muscle cells, including upregulation of oxidative phosphorylation (OXPHOS), cholesterol efflux, collagen, and pro-survival genes while downregulating inflammatory pathways. In vitro studies using bone marrow derived macrophages confirmed AL002a’s ability to drive cell survival, proliferation, OXPHOS, and cholesterol efflux in culture. Finally, we determined that AL002a’s ability to reshape aspects of foamy macrophage function was dependent on its ability to activate the kinase SYK downstream of Trem2. Overall, this data identifies Trem2 as a viable therapeutic target for promoting atherosclerotic plaque stability and improving outcomes in CVD.

Collagen peptides affect collagen synthesis and the expression of collagen, elastin, and versican genes in cultured human dermal fibroblasts.

Collagen is one of the major proteins of the skin and it is particularly important for its strength and resilience. Skin aging is a natural process that is characterized by the decrease and fragmentation of collagen in the dermis. Oral supplementation with collagen peptides has been clinically shown to have a positive effect on the skin condition. However, the mechanisms of aging-related changes synthesized by cells exposed to collagen are currently not well understood. Therefore, in this in vitro study, the mechanisms associated with collagen, elastin, and versican in human dermal fibroblasts were investigated after exposure to collagen peptides. The effects of different concentrations of collagen peptides on cell viability and metabolism were analyzed. For gene expression analysis, human dermal fibroblasts were treated with collagen peptides. This was then followed by RNA extraction and DNA synthesis. Gene expressions of collagen type 1 (COL1A1), elastin (ELN), and versican (VCAN) were quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). In addition, collagen levels were analyzed by confocal scanning laser microscopy using immunostaining. Collagen peptides tested in the study increased the expression of the relevant COL1A1, ELN, and VCAN genes in human dermal fibroblasts (p < 0.005). Furthermore, confocal microscopy showed increased collagen expression in the dermal fibroblast culture after treatment with the collagen peptides (p < 0.005). These data provide cell-based evidence for the beneficial effects of exposure to collagen peptides on the skin's collagen content and on the molecules that provide firmness and elasticity. This may support the hypothesis that collagen peptides are important for maintaining extracellular matrix (ECM) structure and skin regeneration.

Bioactivity and antibacterial effects of zinc-containing bioactive glass on the surface of zirconia abutments

ObjectivesThis study aimed to enhance gingival fibroblast function and to achieve antibacterial activity around the implant abutment by using a zinc (Zn)-containing bioactive glass (BG) coating. Methods45S5 BG containing 0, 5, and 10 wt.% Zn were coated on zirconia disks. The release of silica and Zn ions in physiological saline and their antibacterial effects were measured. The effects of BG coatings on human gingival fibroblasts (hGFs) were assessed using cytotoxicity assays and by analyzing the gene expression of various genes related to antioxidant enzymes, wound healing, and fibrosis. ResultsBG coatings are capable of continuous degradation and simultaneous ion release. The antibacterial effect of BG coatings increased with the addition of Zn, while the cytotoxicity remained unchanged compared to the group without coatings. BG coating enhances the expression of angiogenesis genes, while the Zn-containing BG enhances the expression of antioxidant genes at an early time point. BG coating enhances the expression of collagen genes at later time points. ConclusionsThe antibacterial effect of BG improved with the increase in Zn concentration, without inducing cytotoxicity. BG coating enhances the expression of angiogenesis genes, and Zn-containing BG enhances the expression of antioxidant genes at an early time point. BG coating enhances the expression of collagen genes at later time points. Clinical SignificanceAdding 10 wt% Zn to BG could enhance the environment around implant abutments by providing antibacterial, antioxidant, and anti-fibrotic effects, having potential for clinical use.

Characterization of cardiac fibrosis in older lean vs. obese male ZSF1 rats

The ZSF1 rat is a novel strain produced by crossing a ZDF (Zucker diabetic fatty) female and SHHF (Spontaneously Hypertensive Heart Failure) male rat. Offspring of this hybrid have two phenotypes: healthy lean or obese prone to diabetes and heart failure. The metabolic syndrome and hypertension displayed by the obese ZSF1 rat mimics that seen in individuals who are predisposed to heart failure with preserved ejection fraction (HFpEF). Historically, HFpEF is a diffcult disease to accurately model with rodents and the ZSF1 rat is a promising model for the spontaneous progression to HFpEF. In recent studies, the oldest age at which ZSF1 rats were studied was 32 weeks and these obese rats displayed diastolic dysfunction, cardiac fibrosis, and hypertrophy. Since the prevalence of HFpEF increases with advanced age, the aim of the current study is to evaluate the extent of fibrosis in older ZSF1 rats (40-42 weeks) and determine whether this age group is more appropriate for the study of fibrotic cardiac remodeling related to HFpEF. We hypothesize that left ventricular (LV) fibrosis and collagen gene expression will be increased in obese compared to lean counterparts. Furthermore, we expect that the extent of fibrosis in this age group will exceed that reported in 32-week-old rats; thus, providing an improved model for the study of fibrotic heart disease. Lean and obese male ZSF1 rats (n=8/group) were aged to 40-42 weeks. Rats were anesthetized with a ketamine/xylazine cocktail and cardiac function was evaluated by invasive hemodynamics. The heart was removed, and the LV was trimmed and weighed. A mid-myocardial section from the LV was fixed and embedded in paraffn for histology. Remaining LV tissue was frozen for extraction of RNA and protein for biochemical analysis. LV collagen was assessed by Sirius red stain and collagen I ( Col1a1) and III ( Col3a1) gene expression were assessed by RTqPCR. With no significant difference in heart rate, obese ZSF1 rats had higher LV developed pressure compared to lean (191.1 vs. 122.0 mmHg, p&lt;0.05) and increased maximum and minimum dP/dT (p&lt;0.05). Obese ZSF1 rats exhibited cardiac hypertrophy as measured by LV weight normalized to tibial length (30.21 vs. 23.61 mg/mm, p&lt;0.05, n=7/group) and increased fibrosis (Sirius red) compared to lean counterparts. Furthermore, the fibrosis in obese ZSF1 rats was concentrated in the endocardial and perivascular regions, and qualitatively more than previously published in the 32-week-old ZSF1. Preliminarily, obese ZSF1 rats exhibit a 3.5-fold increase in Col1a1 (p=0.02) and a 19-fold increase in C ol3a1 (p=0.06) gene expression compared to lean (n=3/group). Interestingly, not only is this a larger increase than that reported in 32-week-old old ZSF1s, but the relative increase in Col3a1 vs. Col1a1 is larger. Taken together, these data suggest a progression of cardiac fibrosis beyond what has been reported in the 32-week-old ZSF1 rats. Use of aged ZSF1 rats may be valuable for the study of fibrogenic mediators in HFpEF for the elucidation of therapeutic agents to reduce or reverse cardiac fibrosis. Future studies will include female rats to evaluate sex-specific progression of fibrosis. North Carolina Biotechnology Center Flash Grant (JH) and ECU REDE startup program (AG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prohibitin 1 deletion from activated cardiac fibroblasts in vivo attenuates cardiac fibrosis

Cardiac fibroblasts (CF) are responsible for extracellular matrix (ECM) deposition with cardiac injury. While fibrosis is a typical response to injury, excessive or uncontrolled fibrotic remodeling by CFs underlies the progression to heart failure. Cardiac injury-induced activated CFs are characteristically identified as periostin expressing cells that are proliferative and ECM secreting. This activated secretory phenotype is supported by altered mitochondrial mechanisms. Prohibitin 1 (PHB1) is known to play a role in cell survival via its impact on mitochondrial integrity, and we have established that it is enriched in activated CFs. This study aims to investigate the impact of conditional PHB1 deletion in vivo from activated CFs, and it is expected that PHB1 deletion will reduce or prevent isoproterenol (ISO)-induced fibrotic remodeling. Male and female Periostin-Cre (PC) Phb1fl/fl and WT Phb1fl/fl mice were infused with ISO (75 mg/kg/day) or vehicle (0.003% ascorbic acid in saline) for 1 or 2 weeks via osmotic micro-pump. Cre recombinase was induced by tamoxifen injection (75 mg/kg, i.p.). Hearts were collected, a mid-myocardial section was fixed and stained for fibrosis (Sirius red), and remaining tissue was frozen for protein and RNA extraction. Two weeks of ISO tended to increase hypertrophy in male (p=0.06) and female (p=0.08) mice compared to vehicle. Periostin protein expression was modulated similarly in male and female hearts, which exhibited a significant 7-fold increase following 2 weeks of ISO infusion (p=0.003 vs. vehicle). CF PHB1 deletion significantly attenuated this increase (p=0.009 vs. WT ISO). PHB1 deletion in activated CFs resulted in reduced Col1a1 gene expression following 2 weeks of ISO in males (38.6%) and in females (68.0%) relative to WT ISO. Further, both male and female PC mice exhibited ~55% reduction in Col3a1 gene expression after ISO infusion compared to WT. Taken together, these findings indicate that PHB1 deletion prevents ISO-induced CF activation in both sexes. Left ventricular fibrosis via Sirius red stain was more pronounced in mice treated for 2 weeks compared to 1 week, with males having markedly more fibrosis than females. The accumulation of ISO-induced fibrosis in males was prevented by PHB1 deletion. Interestingly, the extent of cardiac fibrosis with and without PHB1 deletion correlated with periostin protein expression in males, but not in females. Thus, the measured reduction in CF activation by conditional PHB1 deletion may limit ECM deposition in males. The lack of fibrotic tissue in the presence of elevated collagen gene expression and periostin in the ISO-treated females may reflect sex-specific CF phenotypes or fibrotic remodeling. In this model of fibrosis, it is unclear how PHB1 deletion is impacting the female heart structurally. Future studies will aim to elucidate altered mitochondrial mechanisms after PHB1 deletion in models of fibrosis, as well as sex-specific mechanisms of fibrotic remodeling. ECU REDE startup program, NIH T32HL007249-44, UArizona Sarver Heart Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-Specific Regulation of Local Gonadal Hormone Signaling During Pathological Cardiac Remodeling in Spontaneously Hypertensive Rats

Hypertensive heart disease is characterized by cardiac fibrosis in the left ventricle (LV). Angiotensin II (Ang II) promotes cardiac fibrosis through direct actions on cardiac fibroblasts. Gonadal hormones are known to impact cardiac fibrosis either through direct actions on fibroblasts or through modulation of Ang II receptors. We have previously shown that Ang II infusion alters collagen gene expression in a sex-specific manner. The present study seeks to further understand our observed sex difference by investigating the impact of Ang II on gonadal hormone regulation in the left ventricle (LV) and cardiac fibroblasts (CFs) following a fibrogenic stimulus. For whole LV analysis, male and female SHR (15-week-old) were infused with Ang II (400ng/kg/min, s.c.) or vehicle for 2 weeks via an osmotic mini pump (n=8-11 per group per sex). For CF-specific analysis, primary CFs were isolated from adult male and female SHR LV and cultured on CytoSoft cell culture plates (8 kPa stiffness) coated with Type I Collagen. CFs were brought to P1 for expansion and treated with TGFb1 (10ug/uL) or vehicle (PBS + 0.1% BSA) for 48 hours (n=7-8 per group per sex). Col1a1, AT1aR, estrogen receptors α and β (ER-α, ER-β), androgen receptor (AR), 5α-reductase (5α-R), and aromatase gene expression in the left ventricle was assessed via RT-qPCR and data analyzed by Two-Way ANOVA. Ang II infusion significantly increased ER-α gene expression in male, but not female LV (sex x Ang II interaction p=0.0303). ER-β gene expression was also significantly increased in the male LV (p=0.0066), but not females. AR gene expression remained unchanged in the Ang II-infused male LV but was significantly reduced in female LV when compared to control (sex x Ang II interaction p=0.0084). Ang II significantly increased 5α-R gene expression in male (p=0.0024), but not female LV. Aromatase gene expression tended to increase in both male and female Ang II-infused animals when compared to control. Ang II-induced changes in LV gonadal hormone gene expression did not significantly correlate with Col1a1 gene expression. In vitro stimulation of CFs with TGFb1 results in a significant reduction in ER-α (p&lt;0.0001) and AR (p&lt;0.0001) in both males and females. ER-β tended to decrease in males stimulated with TGFb1, but not females, relative to control. Taken together, the opposing findings between LV and CF may indicate cell-specific regulation of gonadal hormone receptors. It may be that upregulation of local estrogen signaling in the remodeling LV in males serves to offset the pathological effects of Ang II stimulation. Future studies will evaluate the consequences of changes in gonadal hormone receptor levels at the cellular and tissue level. NHLBI R01HL153112. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of the safety and efficacy of cosmetics ingredient Spherulites Paeony Superior Retinol.

Microencapsulation of hydroxypinacolone retinoate (HPR) can improve its application in cosmetics. To investigate the safety and efficacy of Spherulites Paeony Superior Retinol, a HPR microcapsule containing 5%-10% peony seed oil, 0.01%-1% epigallocatechin gallatyl glucoside (ECGG), and 0.1%-1% HPR. The safety of Spherulites Paenoy Superior Retinol was evaluated with zebrafish embryo self-rotation irritation test and developmental toxicity test. SymRenew™ HPR was used as a reference. The skin care efficacies of Spherulites Paenoy Superior Retinol were evaluated using zebrafish embryos covering antioxidation, anti-inflammation, blood circulation, whitening, wound healing, skin barrier protection, Type I collagen, elastin, and 5α-reductase genes expression activities. The irritation test revealed that 250 μg/mL Spherulites Paenoy Superior Retinol did not, while 20 μg/mL SymRenew™ HPR significantly (p < 0.05) increased zebrafish embryo self-rotation frequency. The developmental toxicity test found the teratogenicity index (half lethal concentration/half toxicity concentration) of Spherulites Paenoy Superior Retinol and SymRenew™ HPR were 1.9 and 3.1, respectively. The efficacy analysis results showed that 5 μg/mL Spherulites Paenoy Superior Retinol significantly (p < 0.05) exerted 7.1% anti-ROS, 20% anti-inflammation, 14% enhanced blood circulation, 10% suppressed melanin synthesis, 9% enhanced tail fin regeneration, 72% elicited skin barrier protection activity, enhanced the expression of Type I collagen genes col1a1, col1a2, and col1a2 by 34%, 51%, and 42%, respectively, and elastin gene elna by 46%, and suppressed the expression of 5α-reductase genes srd5a1, srd5a2a, and srd5a2b by 52%, 15%, and 30%, respectively. This study demonstrated that Spherulites Paenoy Superior Retinol is a safe cosmetic ingredient with multi-skin care efficacies.

E052 Characterising the factors associated with response to pain management in patients with the Stickler syndrome

Abstract Background/Aims The Stickler syndrome is associated with functional changes in one of the COL2 (type 1, common), COL11 (type 2, uncommon) or COL9 (very rare) genes and causes early onset osteoarthritis. Previous studies have looked at the factors associated with response to pain management interventions in osteoarthritis, but none have done so in a Stickler syndrome cohort. We aimed to identify the factors associated with response to a pain management clinic in patients with osteoarthritis caused by Stickler syndrome. Methods All attendees completed the Brief Pain Inventory (BPI). Scores from 321 Stickler syndrome patients were gathered during each attendance of clinic. Repeat attenders were categorised into responders (BPI score improved on average by &amp;gt; 2 points) and non-responders (BPI score worsened on average by &amp;gt; 2 points). The responders (R) and non-responders (NR) cohorts were characterised and compared using variables including initial pain scores, pain management used, pain categories affected, time between visits, sex, age, genetic mutations, pain location and presence of joint replacements. Results 237/321 patients re-attended the clinic. 26/237 were Responders (R) and 30/237 were Non-responders (NR). All baseline BPI scores were statistically significantly higher in the R cohort, suggesting that the clinic provides greater benefit to those with greater levels of pain. Patients in the total cohort who started with high pain scores (7 or above) had an improvement in scores compared to those who started with low pain scores (3 or below), who had a worsening of scores (-1.158 vs 0.8194, p &amp;lt; 0.0001). Furthermore, there was a weak negative correlation between initial pain score and change in score (r2=0.136). Topical NSAIDs were more commonly used in the NR cohort (33.33% in NR vs 7.69% in R, p = 0.0248) whereas oral NSAIDs were more commonly used in the R cohort (6.67% in NR vs 30.77% in R, p = 0.0332). A final difference between the cohorts was the effect of the pain on the different interference scores. The NR cohort had a statistically significantly lower ‘relations’ score compared to the two most affected interference categories (p = 0.0113, p = 0.0214), whereas the R cohort had no statistically significant difference between the highest affected categories and ‘relations’, suggesting ‘relations’ were disproportionally unaffected in the NR cohort. The time between visits, sex, age, mutations, pain location and joint replacement presence were not statistically significantly different between the two cohorts. Conclusion Patients with high levels of pain benefited from the service. Factors associated with response were high initial pain scores, use of oral NSAIDs, and a ‘relations’ score which was not statistically significantly different to the other life quality categories. This information could be used in the future to help predict response to pain management interventions in Stickler syndrome. Disclosure R. Mudkavi: None. N. Shenker: None.

Precision diagnosis and therapeutic intervention of Alport syndrome

Alport syndrome is one of the most common inherited kidney diseases caused by mutations in the type Ⅳ collagen genes. It has a complex pattern of inheritance and diverse clinical manifestations, and severe cases will rapidly progress to end-stage kidney disease. With the rapid development of genetic testing technology, there is a deeper understanding of the genetic spectrum of Alport syndrome, the effectiveness of clinical therapies, and the prediction of disease prognosis. Therefore, the purpose of the article is to introduce the advances in the diagnosis and treatment of Alport syndrome, aiming to improve the early diagnosis and standardized treatment of this disease.