Small Leucine-rich Proteins Research Articles

Abstract 14979: MicroRNA-342-3p Regulates Post-Ischemic Angiogenesis in Diabetic Mice

Introduction: Diabetic patients are more likely to suffer from adverse cardiovascular events such as myocardial infarction (MI) compared to non-diabetics. Angiogenesis is crucial to limiting infarct size. However, this angiogenic response is blunted in patients with diabetes. MicroRNAs (miRNAs) have been shown to affect both physiological and pathological angiogenesis. However, the role of miRNAs in MI-associated angiogenesis in diabetes remains poorly defined. Here, we describe a novel role of miRNA-342-3p in angiogenesis following ischemic injury to the myocardium. Methods: We performed RNA sequencing on cardiac endothelial cells of Ldlr-deficient mice on either chow or high fat sucrose containing (HFSC) diet at 0-, 3-, 7- and 14-days post-MI. Gain and loss of function studies, functional assays, RNA-seq, bioinformatics, RNA-FISH, and light-sheet microscopy were used to ascertain the function and mechanism of this miRNA. Results: miR-342-3p was upregulated in cardiac ECs during the vascular remodeling phase post-MI in chow-fed mice compared to the HFSC-fed group. Overexpression of miR-342-3p promotes the number of endothelial sprouts (140% vs control), cumulative sprout length (311% vs control) and scratch closure (AUC 83% vs control, p <0.0001) in endothelial cells (ECs), whereas its inhibition had anti-angiogenic effects. Capillary density and EC proliferation (ki67 + /CD31 + ) increased by 107% and154% respectively in the border zone of hearts injected with miR-342-3p mimics compared to control in HFSC diet fed Ldlr -/- mice 7 days after permanent LAD ligation. Using a combination of RNA-seq and prediction algorithms, Decorin (DCN), a small Leucine rich protein with anti-angiogenic properties, was identified as a target of miR-342-3p. Western blot analysis confirmed significant knockdown of Decorin protein by 94% in HUVECs transfected with miR-342-3p mimics compared to controls. RNA seq profiling of overexpressing ECs implicated the ID1 signaling pathway. Conclusion: These results identify a novel role for miR-342-3p in the regulation of angiogenesis post-MI in diabetes. miR-342-3p overexpression rescues impaired angiogenesis in diabetic hearts via the suppression of Decorin, providing new targets for therapeutic angiogenesis.

Data-independent acquisition and quantification of extracellular matrix from human lung in chronic inflammation-associated carcinomas.

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition (DIA) strategies, and stringent statistical processing to analyze "Tumor" and matched adjacent histologically normal ("Matched Normal") tissues from patients with LSCC. Overall, 1802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as "extracellular." Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the "Tumor" compared to "Matched Normal" tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of "Tumor" and "Matched Normal" tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets.

Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc.

Study DesignProfiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD).PurposeTo evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis.Overview of LiteratureLow back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed.MethodsIVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases.ResultsA total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins.ConclusionsThis study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

The endoplasmic reticulum–resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican

The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.

Facet joint degeneration in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a poorly understood deformity of the thoracolumbar spine which affects the intervertebral discs (IVDs) and the articular facet joints. The knowledge concerning facet joints in this context is very limited, although facet joint degeneration is a known contributor of back pain. In this study, a comprehensive investigation was performed to characterize the facet joint chondrocytes and extracellular matrix within the scoliotic spine. Surgically removed articular facet joint tissues were collected from patients undergoing spinal corrective surgery for AIS deformities, while non‐scoliotic articular facet joint tissues were obtained from cadaveric organ donors. Alterations in cartilage tissue structure were evaluated histologically with safranin‐O fast green and a modified OARSI grading scale. Pro‐inflammatory cytokines, matrix‐degrading proteases, and fragmented matrix molecules associated with cartilage degradation were analyzed by immunohistochemistry and western blotting. Safranin‐O fast green staining revealed that young scoliotic facet joints show clear signs of degeneration with substantial proteoglycan loss, similar to osteoarthritis (OA). The proteoglycan levels were significantly lower than in healthy asymptomatic non‐scoliotic control individuals. In comparison to controls, scoliotic articular facets showed increased cell density, increased expression of the proliferation marker Ki‐67, and higher expression of MMP‐3, MMP‐13, and IL‐1β. Expression and fragmentation of the small leucine‐rich proteins (SLRPs) chondroadherin, decorin, biglycan, lumican, and fibromodulin were analyzed with western blot. Chondroadherin and decorin were fragmented in cartilage from patients with a curve greater than 70°, whereas biglycan and fibromodulin did not show curve‐related fragmentation. AIS facet joint cartilage shows hallmarks of OA including proteoglycan loss, overexpression of pro‐inflammatory mediators, increased synthesis of matrix‐degrading proteases and fragmentation of SLRPs. As with patients with age‐related OA, the premature joint degeneration seen in scoliotic patients is likely to contribute to the pain perceived in some individuals.

Abstract 19387: Extracellular Matrix Remodelling in Porcine Coronary Arteries Upon Stent Implantation

Introduction: Extracellular matrix (ECM) remodeling upon vascular stent injury contributes to both neointimal hyperplasia (restenosis) as well as endothelialization (propensity for thrombosis). Despite its important clinical implications little is known about ECM changes post stent implantation. Hypothesis: ECM remodelling, a determinant of stent healing, differs between drug-eluting (DES) and bare-metal stents (BMS). Methods: Using proteomics, we profile changes of vascular ECM proteins over time following implantation of DES and BMS in porcine coronary arteries in an overstretch model. Results: DES (n=14) or BMS (n=14) were implanted in coronary arteries of pigs. Quantitative coronary angiography and optical coherence tomography were performed at 1, 3, 7, 14 and 28 days after stent deployment. Animals were sacrificed at each of these time-points and their coronary arteries were retrieved for subsequent analysis by proteomics. A total of 126 ECM and ECM-associated proteins were identified by mass spectrometry. Within the first week after stent injury, proteins involved in inflammation and thrombosis were elevated in DES and BMS stented arteries. After one week, significant changes in regulatory ECM proteins such as small leucine rich proteins or matricellular proteins were observed. By day 28, differences between BMS and DES became apparent: basement membrane proteins as markers of cellularity were reduced in DES compared with BMS. In contrast, large aggregating proteoglycans such as aggrecan and versican were increased. Both belong to the lectican protein family that act as linkers of ECM molecules with their highly charged glycan side chains. The proteomics findings were validated by immunoblot analysis and real-time polymerase chain reaction. We were also able to detect aggrecan by immunohistochemistry in clinical specimen confirming differential expression patterns of aggrecan and versican in stented human coronary arteries. Conclusions: In the early course after BMS and DES implantation the ECM changes were similar. Significant differences were identified in the regulation of ECM proteins in long-term follow-up consisting of suppression of basement membrane proteins and up-regulation of large aggregating proteoglycans in DES.

The Novel Small Leucine-rich Protein Chondroadherin-like (CHADL) Is Expressed in Cartilage and Modulates Chondrocyte Differentiation

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.

Inverse relationship between matrix remodeling and lipid metabolism during osteoarthritis progression in the STR/ORT mouse

The biologic changes associated with osteoarthritis (OA) are incompletely understood. The aim of this study was to elucidate the molecular mechanisms underlying OA progression in an STR/Ort murine model of spontaneous disease. Global patterns of gene expression were assessed using microarray analysis of articular cartilage/subchondral bone from the tibial plateaus of STR/Ort mice at 3, 9, and 12 months of age. The age-dependent severity of osteophyte formation and extent of cartilage damage were determined in the corresponding femurs using microfocal computed tomography and the Mankin histologic scoring system. Pathway analysis was used to identify the functions of genes associated with OA progression, and changes in gene expression were confirmed using immunohistochemistry. Six hundred twenty-one genes were associated with both osteophyte formation and cartilage damage in the STR/Ort joints. Genes involved in the development/function of connective tissue and in lipid metabolism were most significantly enriched and regulated during disease progression. Genes directly interacting with peroxisome proliferator-activated receptor alpha (PPARalpha)/PPARgamma were down-regulated, whereas those genes involved with connective tissue remodeling were up-regulated during disease progression. Associations of down-regulation of myotubularin-related phosphatase 1 (a phosphoinositide 3-phosphatase involved in lipid signaling) and up-regulation of biglycan (a member of the small leucine-rich protein family known to modulate osteoblast differentiation and matrix mineralization) with OA progression were confirmed by immunohistochemistry. Since adipogenesis and osteogenesis are inversely related in the developing skeletal tissue, these results suggest that a shift in the differentiation of mesenchymal cells from adipogenesis toward osteogenesis is a component of the OA pathophysiologic processes occurring in the tibial plateau joints of STR/Ort mice.

Growth Plate Zonal Microarray Analysis Shows Upregulation of Extracellular Matrix Genes and Downregulation of Metalloproteinases and Cathepsins following Irradiation

Although the growth plate matrix area fraction increases after irradiation, extracellular matrix (ECM) gene expression in this context has not been studied. The hypothesis was that normally expressed ECM genes would be upregulated after irradiation. The right limbs of six Sprague-Dawley 5-week-old rats were irradiated with the left limbs as controls. Half of the animals were harvested after 1 week and half after 2. Microarray was conducted from normal and irradiated tibial growth plate proliferative zone (PZ) and hypertrophic zone (HZ) chondrocytes separated by laser microdissection at each time point. In situ hybridization (ISH) and real-time polymerase chain reaction (PCR) were used to confirm expression of selected genes. At 1 and 2 weeks after irradiation, both normally expressed ECM genes and others not highly expressed in the normal growth plate showed upregulation. Metalloproteinases and cathepsins were downregulated. PZ gene expression after irradiation exhibited features of the normal HZ, suggesting premature terminal differentiation. ECM genes not highly expressed in the normal growth plate included several members of the small leucine-rich proteins and the ezrin-radixin-moesin family. The effects of irradiation on cathepsin K (Ctsk), integrin binding sialoprotein (Ibsp), and procollagen II alpha 1 (Col2a1), as determined by ISH and real-time PCR, were highly correlated with the microarray results. Accumulation of matrix following radiation injury to the growth plate correlated well with changes in gene expression. Upregulation of genes not normally highly expressed in the noninjured growth plate suggests their importance in the injury and repair response.

APP mRNA splicing is upregulated in the brain of biglycan transgenic mice

Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of β-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB +/− × biglycan +/−) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.

Metabolism and Structure-Function Relationships of Connective Tissue Glycosaminoglycans and Proteoglycans

Connective tissue matrix contains the linear polysaccharide (glycosaminoglycan) hyaluronan, and the glycosaminoglycans chondroitin / dermatan sulfate, and keratan sulfate covalently linked to protein (proteoglycans). Hyaluronan is a linear polymer consisting of alternating N-acetylglucosamine and glucuronic acid residues; chondroitin / dermatan has alternating N-acetylgalactosamine and glucuronic or iduronic acid residues; keratan has galactose and N-acetylglucosamine residues. There are two classes of matrix proteoglycans named “hyalectin” and “SLRP (small leucine-rich protein).” Hyalectins have similar large core proteins and large numbers of glycosaminoglycan chains, while SLRPs have similar small core proteins with few glycosaminoglycan chains. Synthesis proceeds by modification of glucose to form specific sugar nucleotides that act as the direct precusors for forming the glycosaminoglycans and linkage oligosaccharides on specific amino acids of specific proteins. The chondroitin / dermatan and keratan are modified by variable sulfation and in the case of dermatan, by epimerization of some of the glucuronic to iduronic. These modifications provide for multiple structural variations that in turn provide a high degree of specificity in function. In general, the functions of matrix hyaluronan and the matrix proteoglycans relate to interactions with other matrix substances such as collagen to provide scaffolding and shape. Their variable structures provide specificity of function for particular tissues, and specific aspects of interaction with other matrix molecules.

Bone morphogenetic protein‐1 cleaves prodecorin in vitro and in cellulo

Introduction Decorin is a ubiquitously distributed small leucine‐rich protein (SLRP) with pivotal roles in collagen fibrillogenesis, regulation of cell matrix deposition as well as cell growth, adhesion and migration. It is expressed in the proform but found mainly in the processed form in various tissues. However, the role of propeptide of prodecorin is still unclear. A possible function is in the trafficking of prodecorin to specific compartment(s) in the cell where it may bind/interact with other ECM molecules prior to secretion. BMP‐1 is a metalloproteinase that is responsible for the processing of various ECM molecules including procollagens and prolysyloxidase. Probiglycan, a SLRP with 57% homology to prodecorin, has also been shown to be a substrate of BMP‐1, which led to the hypothesis that prodecorin could also be processed by the same enzyme. This would emphasize the important role of BMP‐1 in the deposition of the ECM.Materials and methods To aid detection, Flag‐tag (DYKDDDDK) was introduced into the prodecorin sequence immediately 5′ of the propeptide sequence. This was achieved via a two‐step PCR reaction. The triple‐alanine mutant was generated using the same method replacing the putative BMP‐1 cleavage site EDE (30–32) with AAA residues. Transient and stable transfectants were generated in HT1080 human fibroblast cells. These were chosen because of their ability to express and secrete the mutant prodecorin. Purification of decorin was achieved using anion exchange chromatography. BMP‐1‐Flag was expressed in, and purified from, transfected 293‐EBNA cells.Results BMP‐1 was assayed for cleavage of prodecorin in vitro. Addition of BMP‐1 to the assay resulted in removal of the prodomain. Addition of EDTA restores detection of prodecorin by anti‐Flag antibody. HT1080 cells doubly transfected with Flag‐tagged prodecorin and BMP‐1‐Flag secreted decorin in the mature form. Control cells secreted uncleaved prodecorin. HT1080 cells doubly transfected with Flag‐tagged triple‐alanine prodecorin mutant and BMP‐1‐Flag did not show cleavage of prodecorin. It was observed that the GAG‐chain addition to this mutant was affected by the mutation.Discussion BMP‐1 cleaves prodecorin in vitro and in cellulo. The cleaved bond is possibly with the E‐DE (30–32) sequence, but this will be confirmed by cleavage of prodecorin and sequencing of the mature protein. These residues are also important in the initiation and stability of the glycosaminoglycan chain of decorin, because mutation of these results in production of mainly unglycanated prodecorin. This suggests a possible role of the prodomain in the initiation of the GAG chain.