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Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-β, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice – a phenotype that correlates with the tendon pathology. Introduction Tendon is a fibrous tissue that connects skeletal muscle to bone to facilitate motion, whereas ligaments connect articulating bones to support joint alignment and function (Nourissat et al., 2015; Screen et al., 2015). The extracellular matrix of tendons/ligaments is primarily composed of type I collagen as well as smaller quantities of other collagens and proteoglycans (Kannus, 2000). During development, the collagen fibrils in tendons and ligaments develop through addition and lengthening before transitioning to the appositional fusion of existing fibers with continued lengthening in postnatal life (Kalson et al., 2015). The synthesis and assembly of this collagen-rich matrix are influenced by other minor collagens and proteoglycans as well as by the cross-linking chemistry of type I procollagen fibrils, which in turn regulates fibril size and strength (Saito and Marumo, 2010). Like tendon and ligament, the organic matrix of bone consists largely of type I collagen (Alford et al., 2015), and disruptions in collagen synthesis and folding are known to negatively impact its biochemical and mechanical properties in connective tissue diseases such as Osteogenesis Imperfecta (OI) (Lim et al., 2017a). However, despite evidence of joint mobility phenotypes and motor deficits in OI patients (Arponen et al., 2014; Primorac et al., 2014), tendon and ligament phenotypes in this disease are relatively understudied. OI is a heterogeneous group of disorders characterized by variable short stature, skeletal deformities, low bone mass, and increased bone fragility. Approximately 80% of OI cases are caused by dominantly inherited mutations in the genes encoding the α1(I) or α2(I) chains of type I collagen. Mutations in genes responsible for the synthesis, post-translational modification, and processing of collagen, such as cartilage-associated protein (CRTAP), lead to severe, recessive forms of this disease (Lim et al., 2017a). In addition to skeletal defects, other connective tissue manifestations, including joint hypermobility and skin hyperlaxity, are observed in a subset of OI patients (Arponen et al., 2014; Primorac et al., 2014). Our and others' studies have shown that CRTAP forms a complex with Prolyl 3-hydroxylase 1 (P3h1) and Cyclophilin B (CypB, encoded by Ppib) and is required for prolyl 3-hydroxylation of type I procollagen at Pro986 of chain α1(I) and Pro707 of chain α2(I) (Hudson and Eyre, 2013; Morello et al., 2006; Baldridge et al., 2008). In this regard, loss of either CRTAP or P3H1 leads to loss of this complex and its activity, causing a severe recessive form of OI characterized by short stature and brittle bones (Morello et al., 2006; Barnes et al., 2006; Cabral et al., 2007; van Dijk et al., 2009). Collagen isolated from Crtap-/- and P3h1-/- mice is characterized by lysine over-modifications and abnormal fibril diameter (Morello et al., 2006; Cabral et al., 2007). While a comprehensive analysis of Crtap-/- mice has revealed multiple connective tissue abnormalities, including in bones, lungs, kidneys, and skin (Baldridge et al., 2010; Grafe et al., 2014), the impact of the loss of CRTAP on tendons and ligaments remains unknown. Alterations in collagen fibril size and cross-linking have been noted in a limited number of studies using dominant or recessive mouse models of OI (Chen et al., 2014; Terajima et al., 2016; Vranka et al., 2010); however, whether loss of CRTAP impacts tendon and ligament development and structure remains unknown. In this study, we show that Crtap-/- mice have weaker and thinner Achilles and patellar tendons at 1 and 4 months-of-age that are hypercellular with a reduction in tendon volume – a phenotype absent at postnatal day 10 (P10). Examining the collagen matrix, we found an increase in stable (irreversible) collagen cross-links at both timepoints, accompanied by alterations in fibril diameter at 4-months compared to wildtype controls. RNA-seq analyses revealed both shared and distinct changes in the transcriptome of the Achilles and patellar tendons of Crtap-/- mice compared to wildtype with a predicted activation of transforming growth factor-β (TGF-β) and inflammatory signaling in both tissues. Changes in gene expression assessed by qRT-PCR revealed an upregulation of several tendon markers in Crtap-/- Achilles tendons at 1-month, including scleraxis (Scx), type I collagen α1 chain (Col1a1), lumican (Lum), tenascin-C (Tnc), and tenomodulin (Tnmd). At the same time, markers of vascularization (i.e., CD31), and collagen extracellular matrix (ECM) (i.e., type I collagen α2 chain (Col1a2), type II collagen α1 chain (Col2a1), type III collagen α1 chain (Col3a1), type IX collagen α2 chain (Col9a2)) were not different between groups. Many of these changes were also observed in our RNA-seq experiments. Consistent with the gene expression data at 1-month, 4-month-old Crtap-/- mice showed increased α smooth muscle actin (αSMA), matrix metalloproteinase-2 (MMP2), and phospho-nuclear factor kappa B (NFκB) in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. These changes in Crtap-/- tendons were accompanied by motor deficits and reduced strength at 4 months-of-age. In conclusion, loss of CRTAP in mice causes abnormalities in load-bearing tendons and significant behavioral impairments. Results Tendon structure, size, and strength are altered in Crtap-/- mice at 1- and 4-months Mice lacking CRTAP present with growth delay, rhizomelia, and severe osteoporosis together with disruption of other connective tissues, including lung and skin (Morello et al., 2006; Baldridge et al., 2010). To assess abnormalities in the load-bearing tendons from Crtap-/- mice, we harvested ankle and knee joints at 1 and 4 months-of-age to histologically examine the Achilles and patellar tendons. At 1-month, Crtap-/- mice presented with thinner Achilles and patellar tendons (Figure 1C,F) compared to wildtype (Figure 1A,D) and heterozygous mice (Figure 1B,E) with increased cell density in both structures (Figure 1M). By 4 months-of-age, Crtap-/- Achilles and patellar tendons remained thinner and hypercellular compared to wildtype and heterozygous mice (Figure 1G–L,N). Interestingly, ectopic chondrogenesis was present towards either end of the patellar tendon in some (but not all) 4-month-old Crtap-/- mice (Figure 1L) – a phenomenon that can occur in tendinopathy (Steinmann et al., 2020). Consistent with our histological data, phase-contrast µCT analysis demonstrated that patellar tendon volume was reduced in Crtap-/-, but not in heterozygous mice, compared to wildtype controls (Figure 1O–Q). In contrast, no significant changes in articular cartilage volume or surface were observed (Figure 1R,S). Figure 1 Download asset Open asset Loss of CRTAP causes thinning, hypercellularity, and weakening of tendons in young and mature mice. (A–C) Representative H & E images of 1-month ankle joints. (D–F) Representative H & E images of 1-month knee joints. (G–I) Representative H & E images of 4-month ankle joints. (J–L) Representative H & E images of 4-month knee joints. For all micrographs, higher magnification images of the mid-tendon are illustrated. n = 3–4 mice per group. Scale bar is 1 mm. (M–N) Quantification of cell density for Achilles and patellar tendons of wildtype and Crtap-/- mice at 1 month (M) and 4 months (N) of age. Data are min-to-max box and whisker plots with individual points indicated. n = 3 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using two-tailed unpaired t-tests. Exact p-values are reported. (O–P) Representative phase-contrast μCT images of 4-month wildtype (O) and Crtap-/- (P) knee joints. Blue indicates the patellar tendon, green indicates the femoral articular cartilage, and red indicates the tibial articular cartilage. Scale bar is 1 mm. (Q) Quantification of the patellar tendon volume in wildtype, heterozygous, and Crtap-/- mice at 4-months. Data are min-to-max box and whisker plots with individual points indicated. n = 3 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using one-way ANOVA with Tukey's post-hoc tests. Exact p-values are reported. (R–S) Quantification of articular cartilage volume (R) and surface (S) in wildtype, heterozygous, and Crtap-/- mice at 4-months. Data are min-to-max box and whisker plots with individual points indicated. n = 5 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using one-way ANOVA with Tukey's post-hoc tests. Exact p-values are reported. (T–U) Biomechanical assessment of ultimate load (T) and linear stiffness (U) for Achilles tendons from 1-month-old wildtype, heterozygous, and Crtap-/- mice. Data are min-to-max box and whisker plots with individual points indicated. n = 3–8 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using one-way ANOVA with Tukey's post-hoc tests. Exact p-values are reported. To determine whether the altered structure of Crtap-/- load-bearing tendons results in reduced tissue strength, we performed biomechanical testing at 1-month on Achilles tendons to examine structural properties. As predicted based on the histological data, we observed decreases in ultimate load and linear stiffness for Crtap-/- Achilles tendons compared to heterozygous and wildtype mice (Figure 1T,U). Taken together, load-bearing tendons in Crtap-/- mice present with reduced size, increased cell density and decreased strength compared to controls. Given Crtap is deleted throughout development in our global knockout mouse model, we next examined changes in the Achilles and patellar tendons at postnatal day 10. Interestingly, the Crtap-/- Achilles tendons (Figure 2A,B) but not the patellar tendons (Figure 2C,D) were slightly thinner (although not significant) compared to wildtype mice (Figure 2F), with no differences in cell density seen in either tissue (Figure 2E). Taken together, this data suggests that the tendon phenotypes observed at 1-month and beyond occur postnatally and are not due to defects in tendon development. Figure 2 Download asset Open asset Tendon thinning and hypercellularity are not observed in Crtap-/- mice at postnatal day 10. (A–B) Representative H & E images of postnatal day 10 (P10) ankle joints. (C–D) Representative H & E images of P10 knee joints. For all micrographs, higher magnification images of the mid-tendon are illustrated. n = 5 mice per group. Scale bar is 0.5 mm. (E) Quantification of cell density and (F) Average tissue area for Achilles and patellar tendons of wildtype and Crtap-/- mice at P10 (taken mid-tendon). Data are min-to-max box and whisker plots with individual points indicated. n = 5 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using two-tailed unpaired t-tests. Exact p-values are reported. Given the significant hypercellularity seen in the load-bearing tendons from Crtap-/- mice, we next examined whether there were changes in tenocyte populations associated with progenitors and tendon repair response in Crtap-/- mice. Specifically, previous literature has demonstrated that progenitor-like cells involved in tendon maturation and repair are marked by the expression of CD146 (Lee et al., 2015) in addition to others. Using fluorescence-activated cell sorting (FACS) analysis of 5-month-old patellar tendons, we observed a significant decrease in the percentage of CD45-CD31-CD146+CD200+ (~2%) compared to wildtype mice (~4%) (Figure 3A–C, red box). In contrast, CD45-CD31-CD146-CD200+ cells were concomitantly increased in Crtap-/- mice (Figure 3D). Taken together, this data suggests that the matrix disruptions caused by loss of CRTAP may lead to the dysregulation of discrete tendon cell populations within the adult patellar tendon. Figure 3 Download asset Open asset Loss of CRTAP in the patellar tendon leads to a decrease in progenitor cells and an accumulation of immature resident tissue cells. (A–B) Patellar tendon cells isolated from 5-month-old wildtype (A) or Crtap-/- (B) mice were analyzed for the expression of CD200 and CD146 tendon progenitor markers (top histogram) within the CD45-CD31- population (bottom histogram). The plots are representative from a single wildtype or Crtap-/- mouse. (C–D) Graphs show the percentage of CD45-CD31-CD146+CD200+ progenitor cells (C) and CD45-CD31-CD146-CD200+ immature tendon cells (D) From 5-month wildtype and Crtap-/- patellar tendons. Data are min-to-max box and whisker plots with individual points indicated. n = 5 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using two-tailed unpaired t-tests. Exact p-values are reported. Collagen fibril formation is altered in heterozygous and Crtap-/- mice Tendons develop embryonically by increasing in fibril length and number, whereas postnatal growth arises from an increase in fibril length and diameter – the latter of which is driven by the lateral fusion of smaller fibrils (Kalson et al., 2015). To investigate the role of CRTAP in postnatal collagen fibril maturation, we utilized transmission electron microscopy (TEM) to examine changes in fibril diameter in flexor digitorum longus (FDL), Achilles, and patellar tendons (Figure 4). In the FDL tendon, there was a marked increase in small collagen fibrils (20–60 nm in size), a reduction in 80–320 nm fibrils, and a slight increase in larger fibrils (>340 nm in diameter) in Crtap-/- mice compared to wildtype (Figure 4A,C,J). Despite similarities seen in histology, heterozygous mutant FDL tendons also exhibited a slight increase in 20–40 nm fibrils in mice compared to wildtype controls (Figure 4A–B,J). Similar trends were observed for the Achilles tendon, namely an increase in small fibrils (20–60 nm), a reduction in 80–240 nm fibrils, and an increase in large fibrils (>280 nm) upon loss of Crtap (Figure 4D,F,K). In contrast to what we observed for the FDL, heterozygous Achilles tendons did not have increased numbers of smaller fibers (Figure 4E,K). Instead, a greater number of fibrils ranging from 140-to-200 nm in size were noted compared to wildtype controls. Figure 4 Download asset Open asset Collagen fibril diameter is altered in tendons from heterozygous and Crtap-/- mice. (A–C) Representative transverse TEM images of 4-month (A–C) FDL tendon collagen fibrils, (D–F) Achilles tendon collagen fibrils, and (G–I) patellar tendon collagen fibrils. Scale bar is 500 nm. (J) Representative histogram of the size distribution for collagen fibrils in FDL tendons. Data are representative of n = 3 mice. (K) Representative histogram of the size distribution for collagen fibrils in Achilles tendons. Data are representative of n = 3 mice. (L) Representative histogram of the size distribution for collagen fibrils in patellar tendons. Data are representative of n = 3 mice per group. (M–R) Representative longitudinal TEM images of 4-month wildtype FDL (M), Achilles (N), and patellar (O) tendons, and 4-month Crtap-/- FDL (P), Achilles (Q), and patellar (R) tendons. Scale bar is 500 nm. Compared to the FDL and Achilles tendons, the most significant differences were seen within the patellar tendon, although the pattern of changes remained consistent (Figure 4G–I,L). Specifically, we observed a dramatic increase in 20 nm collagen fibrils compared to heterozygous and wildtype mice (Figure 4G–I,L). Fibrils ranging from 100-to-180 nm in diameter were reduced in heterozygous and Crtap-/- mice compared to wildtype. Interestingly, the greatest difference from wildtype was an increase in large collagen fibrils (>200 nm) in both heterozygous and Crtap-/- mice (Figure 4G–I,L). To examine how collagen fibril alignment is affected by the loss of CRTAP, we examined longitudinal sections of FDL, Achilles, and patellar tendons from 4-month wildtype and Crtap-/- mice using TEM (Figure 4M–R). Consistent with our transverse data, we observed a wider array of thinner and thicker collagen fibrils in Crtap-/- tendons (Figure 4P,Q,R) compared to wildtype (Figure 4M,N,O). In addition, while the collagen fibrils in wildtype animals were well-aligned, collagen fibril alignment in Crtap-/- tendons was more irregular (Figure 4P,Q,R). Taken together, these data indicate that loss of CRTAP alters collagen fibril assembly and alignment in load-bearing tendons. In addition, the degree to which collagen assembly is affected is site-dependent. Collagen cross-linking is increased in heterozygous and Crtap-/- mice Along with P3H1 and CyPB, CRTAP is an integral part of the collagen prolyl 3-hydroxylation complex responsible for the 3-hydroxylation of Pro986 of the type I procollagen α1 chain (Lim et al., 2017a). Loss of this complex blocks 3-hydroxyproline formation and affects lysine hydroxylation and cross-linking in bone collagen (Morello et al., 2006; Baldridge et al., 2008); however, whether Crtap-/- tendons display altered collagen cross-linking is unknown. To investigate this, we harvested tendons at 1- and 4-months and assessed collagen cross-linking by quantifying the levels of hydroxylysyl-pyridinoline (HP) (Figure 5). Overall, we observed an increase in these stable, mature collagen cross-links from 1 to 4 months-of-age in all genotypes for the FDL and Achilles tendons (Figure 5A–B). In contrast, for the patellar tendon, age-dependent increases in collagen cross-links were only observed in Crtap-/- mice (Figure 5C). For FDL tendons, Crtap-/- mice had more of these collagen cross-links at 1- and 4-months compared to heterozygous and wildtype mice; however, the content of HP residues per collagen decreased with age in this tissue (Figure 5A). Interestingly, in Achilles tendons, an increase in collagen cross-linking was observed in both heterozygous and Crtap-/- mice at 1-month compared to wildtype. In contrast, at 4-months, only Crtap-/- mice had elevated collagen cross-links, and these levels were greater than those observed at the earlier time point (Figure 5B). Figure 5 Download asset Open asset Collagen cross-linking is increased in tendons from young and mature Crtap-/- mice. Quantification of collagen cross-links as hydroxylysyl-pyridinoline (HP) residues per collagen molecule for (A) FDL tendons; (B) Achilles tendons; and (C) patellar tendons. Data are shown as means ± S.D. n = 3–4 mice per group. Data passed the Shapiro-Wilk test for normality, and groups were compared using one-way ANOVA with Tukey's post-hoc tests. Exact p-values are reported. The patellar tendon showed the greatest increase in collagen cross-links both with time and across genotypes of the tissues examined. Specifically, collagen cross-links were elevated by 5- to 10-fold in Crtap-/- patellar tendons compared to heterozygous and wildtype at 1- and 4-months, respectively (Figure 5C). Taken together, these data suggest that CRTAP is required for proper hydroxylation and cross-linking of collagen fibrils in tendons in a semi-dominant fashion, as heterozygous mutant tendons display a phenotype that is milder than the phenotype observed for homozygous mutant mice. Notably, the chemical quality of collagen cross-linking appears to be spatiotemporally regulated, and this regulation is differentially affected by the loss of a single or both copies of Crtap. Signaling and metabolic dysregulation in Crtap-/- load-bearing tendons We performed bulk RNA-seq with RNA isolated from Achilles and patellar tendons of 1-month-old wildtype and Crtap-/- mice to investigate the molecular changes underlying the observed tendon phenotypes. To determine global changes in differentially expressed genes and predicted upstream regulators, we performed Ingenuity Pathway Analysis (IPA, Qiagen, Germany). For the Achilles tendon, a total of 178 genes (consisting of 99 upregulated genes and 79 downregulated genes) were significantly differentially expressed between wildtype and Crtap-/- samples (Figure 6A). Of the top 30 differentially expressed genes, several ECM proteins, including matrilin-3 (Matn3), matrilin-4 (Matn4), and fibronectin 1 (Fn1), and proteolytic enzymes such as matrix metallopeptidas-2 (Mmp2) were dysregulated. Gene ontology analysis revealed 'GO:000715 – Cell Adhesion', 'GO:0045778 – Positive Regulation of Ossification', and 'GO:0051928 – Positive Regulation of Calcium Ion Transport' to be enriched (Figure 6B). Examination of upstream regulators based on the differential gene expression data revealed a predicted activation of TGF-β1 in Crtap-/- mice and predicted inhibition of dystrophin (DMD) along with several for which activation state was unclear, including platelet-derived growth factor-BB (PDGF-BB), β-catenin (CTNNB1), and tumor necrosis factor (TNF) (Figure 6C). Figure 6 Download asset Open asset Transcriptome analysis of tendons from 1-month-old Crtap-/- mice. DESeq2 was used to compare gene expression between wildtype and Crtap-/- Achilles and patellar tendon RNA samples, and genes with an adjusted p-value<0.05 and absolute log2 fold change >1 were considered as differentially expressed. (A) A bi-clustering heatmap of the top 30 differentially expressed genes between wildtype and Crtap-/- Achilles tendons sorted by adjusted p-value and plotted according to log2 transformed expression values. The Wald test was used to generate p-values and log2 fold changes. See Figure 6—source data 1 for a complete list of differentially regulated genes. (B) Significantly differentially expressed genes between Achilles tendons from wildtype and Crtap-/- mice were clustered by their gene ontology, and enrichment for gene ontology terms was tested using Fisher exact test. All gene ontology terms with an adjusted p-value<0.05 are plotted according to their -log10 adjusted p-value. (C) Select upstream regulators predicted as being activated (shown in green), inhibited (shown in red), or of unclear state (shown in gray) in Crtap-/- compared to wildtype Achilles tendons plotted according to their -log10 adjusted p-value. See Figure 6—source data 2 for a complete list of predicted upstream regulators. n = 3 mice per genotype for (A–C). (D) A bi-clustering heatmap of the top 30 differentially expressed genes between wildtype and Crtap-/- patellar tendons sorted by adjusted p-value and plotted according to log2 transformed expression values. The Wald test was used to generate p-values and log2 fold changes. See Figure 6—source data 3 for a complete list of differentially regulated genes. (E) Significantly differentially expressed genes between patellar tendons from wildtype and Crtap-/- mice were clustered by their gene ontology, and enrichment for gene ontology terms was tested using Fisher exact test. All gene ontology terms with an adjusted p-value<0.05 are plotted according to their -log10 adjusted p-value. (F) Select upstream regulators predicted as being activated (shown in green), inhibited (shown in red), or of unclear state (shown in gray) in Crtap-/- compared to wildtype patellar tendons plotted according to -log10 adjusted p-value. See Figure 6—source data 4 for a complete list of predicted upstream regulators. n = 2 mice per genotype for (D–F). Figure 6—source data 1 List of differentially expressed genes between 1-month wildtype and Crtap knockout Achilles tendons. The Wald test was used to generate p-values and log2 fold changes. with an adjusted p-value<0.05 and absolute log2 fold change >1 were significantly differentially expressed genes. is a list of all differentially expressed genes by adjusted p-value. Download Figure 6—source data 2 upstream regulators differential gene expression between 1-month wildtype and Crtap knockout Achilles tendons. on a list of differentially expressed genes, upstream regulators were predicted using Ingenuity Pathway The results are sorted by adjusted p-value. Download Figure 6—source data 3 List of differentially expressed genes between 1-month wildtype and Crtap knockout patellar tendons. The Wald test was used to generate p-values and log2 fold changes. with an adjusted p-value<0.05 and absolute log2 fold change >1 were significantly differentially expressed genes. is a list of all differentially expressed genes by adjusted p-value. Download Figure 6—source data 4 upstream regulators differential gene expression between 1-month wildtype and Crtap knockout patellar tendons. on a list of differentially expressed genes, upstream regulators were predicted using Ingenuity Pathway The results are sorted by adjusted p-value. Download In with the increased seen in patellar tendons from Crtap-/- mice, a greater number of total genes were differentially expressed between wildtype and Crtap-/- patellar tendons (Figure We significant differential expression of genes, with being upregulated and downregulated in Crtap-/- compared to wildtype. of the top 30 differentially expressed genes were minor collagens such as type IX collagen chain type IX collagen α1 chain and type collagen α1 chain as well as other ECM proteins, including lumican and fibronectin 1 the Achilles tendon, gene ontology analysis revealed significant enrichment for metabolic including – – Cell and – Cell as well as for – Positive Regulation of and – (Figure Examination of upstream regulators also a significant activation of and 6 as well as growth factor 2 and in Crtap-/- patellar tendons (Figure the of TGF-β, and in this is consistent with but more severe than that observed for those same regulators in the Achilles tendon results (Figure 6C). We next examined the expression of genes that are in postnatal tendon maturation and We the loss of Crtap expression in Crtap-/- Achilles tendon compared to wildtype – a also demonstrated in our RNA-seq data data shown in red as fold adjusted with the results in Figure Interestingly, loss of CRTAP to a upregulation in expression with no changes in and levels (Figure Despite a in in Crtap-/- Achilles tendons, there were no changes in the expression of or the other we observed an increase in expression of several ECM and small proteoglycans including and known to be upregulated tendon maturation and We also observed an upregulation in in Crtap-/- Achilles tendons compared to wildtype, consistent with our RNA-seq data (Figure Overall, we that the reduction in expression and altered expression of as well as other ECM such as and be associated with the observed in collagen seen in Crtap-/- mice, and may indicate an increased remodeling or repair response to that seen in OI bone et al., 2014). Figure Download asset Open asset Tendon marker gene expression is altered in Crtap-/- Achilles tendons at 1 was performed to examine changes in the expression of collagens (i.e., tendon other from the RNA-seq

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Introduction: Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic cancer cells undergo an epithelial-to-mesenchymal transition (EMT) regulated by a double-negative feedback loop between the microRNA-200 (miR-200) family and Zeb1, but the precise mechanisms of Zeb1-dependent EMT in promoting malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. This study investigates the collaborative effect of EMT and ECM in the metastatic process. Methods: Bioinformatic analysis of TCGA dataset was done to correlate ECM-associated gene expression with EMT gene signature scores. Western blotting and qPCR analysis of epithelial and mesenchymal lung cancer cell lines were performed to determine expression levels of collagen, LOX, and LOXL2. Lung tumor tissues from non-metastatic KrasG12D and metastatic KrasG12D;p53R172H mutant mice were analyzed by immunohistochemistry, Masson's trichrome staining, and second harmonics generation for collagen, LOX, and LOXL2 expression as well as collagen fiber organization. Syngeneic primary tumors generated by subcutaneous injection of murine lung cancer cell lines were analyzed in a similar fashion. Promoter and 3′-UTR luciferase reporter assays were performed to determine direct regulation LOXL2 and LOX by Zeb1 and miR-200, respectively. Results: Our results reveal increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in Zeb1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and Zeb1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform driving lung cancer metastasis by crosslinking and stabilizing insoluble collagen deposition in primary tumor tissues. Conclusions: Our study demonstrates that mesenchymal lung cancer cells metastasize by modulating the compositional and structural properties of the ECM through LOXL-mediated collagen crosslinking and deposition. We are the first to validate direct regulation of LOX and LOXL2 by the miR-200/Zeb1 axis, delineate collagen as a prognostic marker for lung cancer, and identify LOXL2 as a potential therapeutic target against tumor progression. Citation Format: David H. Peng, Pan Tong, Lauren A. Byers, Jing Wang, Chad J. Creighton, Don L. Gibbons. Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A27.

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Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing.

The molecular weight of chitosan (CS) may affect its physical properties and its ability to induce an appropriate host response. The biocompatibilities of CS membranes of low (LMWCS) and high (HMWCS) molecular weight were investigated by inserting these materials into the subcutaneous tissue of rats for 1-28 days and evaluating leukocyte infiltration, granulation tissue, fibrosis, arginase-1 immunostaining, as well as nuclear factor-κB (NF-κΒ) and fibroblast growth factor (FGF)-2 expressions. Both CS membranes induced a peak of leukocyte infiltration on the first day of insertion and stimulated granulation and fibrous tissue generation when compared to control. LMWCS induced more collagen deposition a week earlier, when compared to the control and HMWCS membrane. The membranes also increased arginase-1 immunostaining, a M2 macrophage marker. M2 macrophage is recognized as anti-inflammatory and pro-regenerative. NF-κB is an essential biomarker of the inflammatory process and induces the expression of several pro-inflammatory cytokines. The LMWCS membrane reduced inflammation, as indicated by a reduced nucleus/cytoplasm NF-κB ratio in surrounding tissue from days 7 to 14 when compared to control. On the first day, the expression of FGF-2, a biomarker of inflammatory resolution, was increased in the tissue of the LWMCS group, when compared with HMWCS, which was consistent with the type I collagen deposition. Thus, LWMCS was associated with a prior reduction of the inflammatory response and improved wound healing.

Topical bilirubin-deferoxamine hastens excisional wound healing by modulating inflammation, oxidative stress, angiogenesis, and collagen deposition in diabetic rats

Adipose-derived stem cells improve full-thickness skin grafts in a rat model

Antibacterial performance and in vivo diabetic wound healing of curcumin loaded gum tragacanth/poly(ε-caprolactone) electrospun nanofibers

Healthy skin protects our bodies from environmental harm. Burns, however, can lead to infections and delayed healing, often with poor cosmetic results. Therefore, promoting efficient wound healing is crucial to restore normal skin function while minimizing scarring. This study investigated the potential benefits of tilapia skin collagen in accelerating burn repair and explored the underlying molecular mechanisms. Analysis using Fourier-transform infrared spectroscopy (FTIR) confirmed that the extracted tilapia collagen (TC) possessed a triple helical structure characteristic of type I collagen. Histopathological examination revealed that TC treatment promoted collagen deposition, epithelization (skin resurfacing), and complete reconstruction of connective tissue compared to the collagen control group (CG). Masson's trichrome staining further supported these findings. The tilapia collagen group (TCG) displayed dense and organized collagen fibers, indicating a more complete remodeling process compared to CG. Notably, TCG exhibited increased expression of growth factors Basic-fibroblast growth factor (b-FGF) and Vascular Endothelial Growth Factor (VEGF), while showing a decrease in Transforming Growth Factor beta 1 (TGF-β1) and alpha Smooth Muscle Actin (α-SMA), suggesting a potential shift towards wound healing and reduced scarring. Topical application of tilapia collagen (TC) appears to promote burn wound healing through several mechanisms. TC may accelerate the healing process by rapidly reducing wound size and increasing the rate of epithelization (skin resurfacing). It may also stimulate collagen production and deposition, leading to a thicker dermis (skin layer). Additionally, TC may promote new blood vessel formation (neovascularization) in granulation tissue, potentially reducing scar formation. Interestingly, studies suggest that collagen interacts with alpha-SMA, but not TGF-β1, potentially explaining the reduced scarring effect. These findings position tilapia-derived collagen as a promising, safe, and cost-effective alternative to existing wound care products.

Objective: To assess the risk factors affecting development of non-tumor- related anastomotic stenosis after rectal cancer and to construct a nomogram prediction model. Methods: This was a retrospective study of data of patients who had undergone excision with one-stage intestinal anastomosis for rectal cancer between January 2003 and September 2018 in Nanfang Hospital of Southern Medical University. The exclusion criteria were as follows: (1) pathological examination of the operative specimen revealed residual tumor on the incision margin of the anastomosis; (2) pathological examination of postoperative colonoscopy specimens revealed tumor recurrence at the anastomotic stenosis, or postoperative imaging evaluation and tumor marker monitoring indicated tumor recurrence; (3) follow-up time <3 months; and (4) simultaneous multiple primary cancers. Univariate analysis using the χ2 or Fisher's exact test was performed to assess the study patients' baseline characteristics and variables such as tumor-related factors and surgical approach (P<0.05). Multivariate analysis using binary logistic regression was then performed to identify independent risk factors for development of non-tumor-related anastomotic stenosis after rectal cancer. Finally, a nomogram model for predicting non-tumor-related anastomotic stenosis after rectal cancer surgery was constructed using R software. The reliability and accuracy of this prediction model was evaluated using internal validation and calculation of the area under the curve of the model's receiver characteristic curve (ROC). Results: The study cohort comprised 1,610 patients, including 1,008 men and 602 women of median age 59 (50, 67) years and median body mass index 22.4 (20.2, 24.5) kg/m². Non-tumor-related anastomotic stenosis developed in 121 (7.5%) of these patients. The incidence of non-tumor-related anastomotic stenosis in patients who had undergone neoadjuvant chemotherapy, neoadjuvant radiotherapy, and surgery alone was 11.2% (10/89), 26.4% (47/178), and 4.8% (64/1,343), respectively. Neoadjuvant treatment (neoadjuvant chemotherapy: OR=2.455, 95%CI: 1.148-5.253, P=0.021; neoadjuvant chemoradiotherapy, OR=3.882, 95%CI: 2.425-6.216, P<0.001), anastomotic leakage (OR=7.960, 95%CI: 4.550-13.926, P<0.001), open laparotomy (OR=3.412, 95%CI: 1.772-6.571, P<0.001), and tumor location (distance of tumor from the anal verge 5-10 cm: OR=2.381, 95%CI:1.227-4.691, P<0.001; distance of tumor from the anal verge <5 cm: OR=5.985,95% CI: 3.039-11.787, P<0.001) were identified as independent risk factors for non-tumor-related anastomotic stenosis. Thereafter, a nomogram prediction model incorporating the four identified risk factors for development of anastomotic stenosis after rectal cancer was developed. The area under the curve of the model ROC was 0.815 (0.773-0.857, P<0.001), and the C-index of the predictive model was 0.815, indicating that the model's calibration curve fitted well with the ideal curve. Conclusion: Non-tumor-related anastomotic stenosis after rectal cancer surgery is significantly associated with neoadjuvant treatment, anastomotic leakage, surgical procedure, and tumor location. A nomogram based on these four factors demonstrated good discrimination and calibration, and would therefore be useful for screening individuals at risk of anastomotic stenosis after rectal cancer surgery.