Abnormal Extracellular Matrix Remodeling Research Articles

340 The Effects of PTSD-Dependent Neurogenic Hypertension and Inflammation on Thoracic Aortic Aneurysm Progression

OBJECTIVES/GOALS: Nearly all thoracic aortic aneurysm patients suffer from hypertension leading to elevated wall tension and abnormal extracellular matrix remodeling. PTSD patients have higher blood pressure both at rest and in response to stimuli. Although stress is associated with cardiovascular disease, the exact mechanism linking the two is still unknown. METHODS/STUDY POPULATION: Adult C57BL/6 mice underwent a PTSD induction protocol consisting of inescapable foot shock followed by single prolonged stress. The mice were assessed incrementally for their PTSD-like phenotype using specific behavioral tests chosen to assess for each of the human criteria of PTSD according to the DSM-V. Tail cuff blood pressure measurements were taken serially throughout the 16-week protocol. At terminal study, thoracic aortic diameter measurements were obtained through digital microscopy and plasma was harvested for cytokine analysis. Thoracic aortic aneurysms (TAA) were induced through periadventitial application of a calcium chloride solution on the descending thoracic aorta in BPH/2J and BPN/3J adult mice. The thoracic aortic diameter was measured at terminal study through digital microscopy. RESULTS/ANTICIPATED RESULTS: Using our PTSD-like mouse model we have demonstrated that PTSD-like mice have significantly higher systolic blood pressure following a reminder of the traumatic event than control mice recapitulating the human phenotype. They also had increased plasma proinflammatory cytokines and larger thoracic aortic diameters than control mice. Although the increased thoracic aortic diameter is not an aneurysm, it suggests ECM remodeling is occurring predisposing the aorta to aneurysm formation. Finally, we have shown that in neurogenic hypertensive mice, TAA formation was accelerated by 12 weeks with roughly 70% dilation at 4 weeks post-TAA induction surgery as compared to roughly a 20% dilation in control mice. DISCUSSION/SIGNIFICANCE: Altogether, these studies reinforce the link between stress and TAA development, and our mouse model will allow for the underlying mechanism to be elucidated. Better understanding of the mechanism linking PTSD and TAA will allow for the creation of novel therapeutics to treat PTSD symptoms while also delaying TAA progression.

Deficiency of Integrin β4 Results in Increased Lung Tissue Stiffness and Responds to Substrate Stiffness via Modulating RhoA Activity.

The interaction between extracellular matrix (ECM) and epithelial cells plays a key role in lung development. Our studies found that mice with conditional integrin β4 (ITGB4) knockout presented lung dysplasia and increased stiffness of lung tissues. In accordance with our previous studies regarding the functions of ITGB4 in bronchial epithelial cells (BECs), we hypothesize that the decreased ITGB4 expression during embryonic stage leads to abnormal ECM remodeling and increased tissue stiffness, thus impairing BECs motility and compromising lung development. In this study, we examined lung tissue stiffness in normal and ITGB4 deficiency mice using Atomic Force Microscopy (AFM), and demonstrated that ITGB4 deficiency resulted in increased lung tissue stiffness. The examination of ECM components collagen, elastin, and lysyl oxidase (LOX) family showed that the expression of type VI collagen, elastin and LOXL4 were significantly elevated in the ITGB4-deficiency mice, compared with those in normal groups. Airway epithelial cell migration and proliferation capacities on normal and stiff substrates were evaluated through video-microscopy and flow cytometry. The morphology of the cytoskeleton was detected by laser confocal microscopy, and RhoA activities were determined by fluorescence resonance energy transfer (FRET) microscopy. The results showed that migration and proliferation of ITGB4 deficiency cells were noticeably inhibited, along decreased cytoskeleton stabilization, and hampered RhoA activity, especially for cells cultured on the stiff substrate. These results suggest that decreased ITGB4 expression results in increased lung tissue stiffness and impairs the adaptation of bronchial epithelial cells to substrate stiffness, which may be related to the occurrence of broncho pulmonary dysplasia.

Transcriptomic and epigenomic analyses uncovered Lrrc15 as a contributing factor to cartilage damage in osteoarthritis

In osteoarthritis (OA), articular chondrocytes display phenotypic and functional changes associated with epigenomic alterations. These changes contribute to the disease progression, which is characterized by dysregulated reparative processes and abnormal extracellular matrix remodeling leading to cartilage degradation. Recent studies using a murine model of posttraumatic OA highlighted the contribution of changes in DNA hydroxymethylation (5hmC) to OA progression. Here, we integrated transcriptomic and epigenomic analyses in cartilage after induction of OA to show that the structural progression of OA is accompanied by early transcriptomic and pronounced DNA methylation (5mC) changes in chondrocytes. These changes accumulate over time and are associated with recapitulation of developmental processes, including cartilage development, chondrocyte hypertrophy, and ossification. Our integrative analyses also uncovered that Lrrc15 is differentially methylated and expressed in OA cartilage, and that it may contribute to the functional and phenotypic alterations of chondrocytes, likely coordinating stress responses and dysregulated extracellular matrix remodeling.

Abstract 122: Pharmacological Inhibition Of Macrophage Infiltration Prevents Myxomatous Valve Degeneration In Marfan Syndrome

Introduction: Myxomatous valve degeneration (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. Currently, there is no medical therapy for the treatment of MVD. MVD in a mouse model of Marfan syndrome (MFS) is characterized leaflet thickening and increased macrophage infiltration, which are reduced with loss of C-C chemokine receptor type 2 (CCR2). However, the specific contributions of macrophages to pathological extracellular matrix (ECM) remodeling and underlying mechanisms are unknown. Hypothesis: Inhibition of macrophage infiltration by a CCR2 inhibitor blocks ECM abnormalities and MVD progression in mitral valves of MFS mice by suppressing the response to cytokine/chemokines. Methods: Mice with the mutation of Fibrillin 1 (Fbn1 C1039G/+ ) recapitulate histopathological features of MFS. Here, we tested the efficacy of a selective CCR2 antagonist RS504393 in the valves of MFS mice in the initiation (1-month-old) and the progression (2-month-old) of MVD, respectively. MFS mice were intraperitoneally injected with RS504393 at 2 mg/kg/d for 30 days. Histological evaluation and immunofluorescence for macrophages and ECM were performed. RNAseq was performed in mitral valves from 2-month old Fbn1 C1039G/+ mice with CCR2 knockout (CCR2 RFP/RFP ). Results: MFS valves revealed ECM abnormalities characterized by collagen fragmentation and proteoglycan accumulation. RS504393 treatment reduced infiltrating macrophages (MHCII+, CCR2+) in myxomatous valves. Remarkably, RS504393 was protective against both the initiation and the progression of MVD, detected by decreased mitral valve thickness and prevention of pathological ECM remodeling in MFS mice. RNAseq data confirmed increased leukocyte activation involved in immune response and abnormal extracellular matrix remodeling in MFS valves. CCR2 deficiency blocked macrophage infiltration and inhibited the response to cytokines in Fbn1 C1039G/+ valves. Conclusions: Our results show that macrophage infiltration is critical for progressive MVD. Moreover, CCR2 inhibition ameliorates MVD progression by preventing immune response. Thus, the CCR2 inhibitor RS504393 is a potential pharmacological candidate to treat MVD in MFS.

Pharmacological Inhibition of Macrophage Infiltration Prevents Myxomatous Valve Degeneration in Marfan Syndrome

Background Myxomatous valve degeneration (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome (MFS) is characterized leaflet thickening and increased macrophage infiltration which are reduced with loss of C-C chemokine receptor type 2 (CCR2). However, the specific contributions of macrophages to pathological extracellular matrix (ECM) remodeling and the underlying mechanisms are unknown. Methods Mice with the mutation of Fibrillin 1 (Fbn1C1039G/+) recapitulate histopathological features of MFS. ECM remodeling in mitral valves was assessed by detecting collagen and proteoglycan in Fbn1C1039G/+; CCR2RFP/RFP mice. Here, we tested the efficacy of a selective CCR2 antagonist RS504393 in the valves of MFS model in the initiation (1-month-old) and the progression (2-month-old) of MVD, respectively. MFS mice were intraperitoneally injected with RS504393 at 2 mg/kg/d for 1 month. Histological evaluation and immunofluorescence for macrophages and ECM were performed. Results MFS valves revealed ECM abnormalities characterized by collagen fragmentation and proteoglycan accumulation. Deficiency of CCR2 inhibited ECM abnormalities in MFS mitral valves. RS504393 treatment reduced infiltrating macrophages (MHCII+, CCR2+) in myxomatous valves. Remarkably, RS504393 was protective against both the initiation and the progression of MVD, detected by decreased mitral valve thickness and prevented pathological ECM remodeling in MFS mice. Conclusion Our results suggest the deficiency of CCR2 inhibits macrophage infiltration and abnormal ECM remodeling. In addition, the CCR2 inhbitor RS504393 is a potential pharmacological candidate to treat MVD in MFS.

Abstract WMP40: Cervical Internal Carotid Artery Tortuosity as a Marker of Non-Atherosclerotic Aging: Results From the Northern Manhattan Study (NOMAS)

Background: Cervical internal carotid artery (ICA) tortuosity has been associated with vascular risk and stroke as well as genetic disorders related to abnormal extracellular matrix remodeling. It is plausible that dystrophic or aberrant arterial remodeling may therefore relate to cervical ICA tortuosity. We hypothesized that cervical ICA tortuosity relates to carotid dilatation, but not to traditional ultrasound (US) markers of atherosclerosis. Methods: Subjects of the NOMAS with available time-of-flight MRA were included in our study. Cervical ICA tortuosity was defined as a bend in the distal cervical ICA of > 90° as seen on MRA. We excluded subjects with < 5 cm of the cervical ICA visualized. Distensibility was calculated as the percentage excursion of the right CCA diastolic diameter during systole, which was assessed by high-resolution B-mode US of the right common carotid artery (CCA). We used multivariable logistic regression analyses to estimate odds ratios for the association of cervical ICA tortuosity and Doppler measures of carotid wall aging. Results: We visualized cervical ICA tortuosity in 468 NOMAS participants (mean age 64±8 years, 70% women, 70% Hispanic). It was present in 23% of subjects. In unadjusted models, cervical ICA tortuosity was more common in women (OR 2.34, 95% CI 1.34-4.11), Hispanics (OR 1.85, 95%CI 1.06-3.25) and those with higher diastolic blood pressures (OR per mm Hg 1.04, 95%CI 1.01-1.06), and less common among smokers (OR 0.23, 95%CI 0.07-0.78). In models adjusted for demographic and vascular risks, right CCA tortuosity was associated with ipsilateral larger CCA DD (OR 1.42, 95%CI 1.02-1.96) and borderline associated with lower distensibility (OR 0.94, 95%CI 0.87-1.01, P=0.06) but not with ipsilateral ICA IMT (OR 0.26, 95%CI 0.14-4.77), number of plaques (OR 1.08, 95%CI 0.76-1.53), maximum plaque thickness (OR 0.96, 95%CI 0.73-1.27), or plaque area (1.00, 95%CI 0.97-1.05). Conclusions: Cervical ICA tortuosity associates with ipsilateral cervical ICA dilatation and lower distensibility, but not with traditional US markers of atherosclerosis. The association with diastolic blood pressure suggests a role of steady, rather than pulsatile, hemodynamics in aberrant cervical ICA remodeling.

ColXV Aggravates Adipocyte Apoptosis by Facilitating Abnormal Extracellular Matrix Remodeling in Mice.

The extracellular matrix (ECM) is a highly dynamic structural network and plays an essential role in cell behavior and regulation during metabolic homeostasis and obesity progression. Abnormal ECM remodeling impairs adipocyte plasticity required for diverse cellular functions. Collagen XV (ColXV) is a proteoglycan localized to the outermost layer of basement membranes (BMs) and forms a bridge between the BMs and the fibrillar collagen matrix. Nevertheless, how ColXV affects ECM composition and the reason for subsequent adipocyte apoptosis is still unclear. This report found, through RNA-seq data, that ColXV is linked to cell growth and ECM remodeling. Findings show that, in response to excessive expression of extracellular ColXV, the AMPK/mTORC1 pathway is strongly activated and triggers a cascade of mitochondrial apoptosis. This is the first study to make use of ECM three-dimensional reconstruction, based on decellularization in the adipose tissues and the study reveals that ColXV is an activation factor that alters ECM remodeling in adipose tissues. It was also demonstrated that the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor 1 (FGFR1) axis involved in ECM remodeling is suppressed by ColXV due to reduction of FGF2 translocation to FGFR1. Furthermore, ColXV induced remodeling of ECM preceding apoptosis and continued to induce apoptosis in adipocytes. Collectively, our findings establish ColXV as a basement membrane collagen with homology to ColXVIII, indicating that it is one of the positive regulators for inducing ECM remodeling and further promoting adipocyte apoptosis.

Remodeling of the interstitial extracellular matrix in white matter multiple sclerosis lesions: Implications for remyelination (failure)

The extracellular matrix (ECM) provides protection, rigidity, and structure toward cells. It consists, among others, of a wide variety of glycoproteins and proteoglycans, which act together to produce a complex and dynamic environment, most relevant in transmembrane events. In the brain, the ECM occupies a notable proportion of its volume and maintains the homeostasis of central nervous system (CNS). In addition, remodeling of the ECM, that is transient changes in ECM proteins regulated by matrix metalloproteinases (MMPs), is an important process that modulates cell behavior upon injury, thereby facilitating recovery. Failure of ECM remodeling plays an important role in the pathogenesis of multiple sclerosis (MS), a neurodegenerative demyelinating disease of the CNS with an inflammatory response against protective myelin sheaths that surround axons. Remyelination of denuded axons improves the neuropathological conditions of MS, but this regeneration process fails over time, leading to chronic disease progression. In this review, we uncover abnormal ECM remodeling in MS lesions by discussing ECM remodeling in experimental demyelination models, that is when remyelination is successful, and compare alterations in ECM components to the ECM composition and MMP expression in the parenchyma of demyelinated MS lesions, that is when remyelination fails. Inter- and intralesional differences in ECM remodeling in the distinct white matter MS lesions are discussed in terms of consequences for oligodendrocyte behavior and remyelination (failure). Hence, the review will aid to understand how abnormal ECM remodeling contributes to remyelination failure in MS lesions and assists in developing therapeutic strategies to promote remyelination.

Roles of microRNAs in abdominal aortic aneurysm pathogenesis and the possibility of their use as biomarkers.

Abdominal aortic aneurysm (AAA) is a dangerous condition involving pathological dilation of the aortic wall. Due to the asymptomatic course of this disease and the dangerous consequences of its rupture, it is important to identify its specific bio-markers expressed as early as possible. Different expression profiles of microRNAs (miRNAs) were detected in patients diagnosed with AAA. MicroRNAs are small non-coding RNA molecules that regulate the expression of other genes at the translation stage. miRNAs affecting translation can lead to abnormal remodeling of extracellular matrix, inhibition of the cell cycle, cell aging or intensified inflammation. This review summarizes current knowledge about the role of microRNAs in the context of formation and development of abdominal aorta aneurysm and the possibility of using some miRNAs as bio-markers, and also provides basic information about miRNAs and aneurysms.

Pathobiochemistry of arthrofibrotic remodeling

Aims and Objectives:Arthrofibrosis is defined as painful impairment of joint flexibility due to fibrotic tissue remodeling after joint trauma or surgery. The incidence of arthrofibrosis after knee replacement surgery is 5 to 10%. Although conventional therapeutic approaches as for instance mobilization and physiotherapy are applied, an effective and causative therapeutic regimen is not known.Materials and Methods:To characterize arthrofibrotic remodeling of the extracellular matrix, to develop new therapeutic approaches and to define diagnostic biomarkers and therapeutic targets, understanding of biochemical principles is urgently required. Fibrotic remodeling was described in several tissues, whereas synovial fibrosis is one of the least investigated fibrotic disorders. Nevertheless, molecular key events in fibrosis seem to be the same and are initiated by exogenic or endogenic tissue damage and differentiation of resident fibroblasts of the connective tissue to myofibroblasts. Known inductors of myofibroblast differentiation are fibrotic growth factors, which are secreted by platelets, damaged tissue and inflammatory cells, as well as mechanical strain. Research studies concerning cardiac fibrosis in tako-tsubo cardiomyopathy also define emotional stress and sympathicotonic destabilization as profibrotic stressors. Myofibroblasts generate contractile forces and synthesize extracellular matrix components, so that scar tissue accumulates. While myofibroblasts disappear by apoptosis in physiological wound healing, they persist in fibrosis.Results:Recently, we could demonstrate that increased expression of human xylosyltransferase (XT)-I, an enzyme which catalyzes the rate limiting step in proteoglycan glycosylation, is linked to abnormal extracellular matrix remodeling. Serum XT activity reflects proteoglycan synthesis rate and is known as fibrosis biomarker in liver fibrosis or scleroderma. Our data also indicate that XT-I is a cellular key mediator of arthrofibrosis. However, we suggest that molecular changes based on arthrofibrosis are, due to local restriction of the affected joint by the blood-synovial-barrier, not detectable in human serum. Currently, we study synovial XT activity of arthrofibrosis patients and controls in a multicenter study.Conclusion:In summary, we give insights into the complex pathobiochemistry of arthrofibrosis as well as current research projects. A deeper characterization of the involved mechanisms might not only contribute to control and inhibit fibrotic remodeling by interfering with components of fibrotic signal cascades but also to establish new therapeutic strategies.

Fernblock (Polypodium leucotomos Extract): Molecular Mechanisms and Pleiotropic Effects in Light-Related Skin Conditions, Photoaging and Skin Cancers, a Review.

Healthier life styles include increased outdoors time practicing sports and walking. This means increased exposure to the sun, leading to higher risk of sunburn, photoaging and skin cancer. In addition to topical barrier products, oral supplementations of various botanicals endowed with antioxidant activity are emerging as novel method of photoprotection. Polypodium leucotomos extract (PL, commercial name Fernblock®, IFC Group, Spain) is a powerful antioxidant due to its high content of phenolic compounds. PL is administered orally, with proven safety, and it can also be used topically. Its mechanisms include inhibition of the generation and release of reactive oxygen species (ROS) by ultraviolet (UV) light. It also prevents UV- and ROS-induced DNA damage with inhibition of AP1 and NF-κB and protection of natural antioxidant enzyme systems. At the cellular level, PL decreases cellular apoptosis and necrosis mediated UV and inhibits abnormal extracellular matrix remodeling. PL reduces inflammation, prevents immunosuppression, activates tumor suppressor p53 and inhibits UV-induced cyclooxygenase-2 (COX-2) enzyme expression. In agreement with increased p53 activity, PL decreased UV radiation-induced cell proliferation. PL also prevents common deletions mitochondrial DNA damage induced by UVA, and MMP-1 expression induced Visible Light and Infrared Radiation. These cellular and molecular effects are reflected in inhibitions of carcinogenesis and photoaging.

AMPK Activation by Metformin Suppresses Abnormal Extracellular Matrix Remodeling in Adipose Tissue and Ameliorates Insulin Resistance in Obesity.

Fibrosis is emerging as a hallmark of metabolically dysregulated white adipose tissue (WAT) in obesity. Although adipose tissue fibrosis impairs adipocyte plasticity, little is known about how aberrant extracellular matrix (ECM) remodeling of WAT is initiated during the development of obesity. Here we show that treatment with the antidiabetic drug metformin inhibits excessive ECM deposition in WAT of ob/ob mice and mice with diet-induced obesity, as evidenced by decreased collagen deposition surrounding adipocytes and expression of fibrotic genes including the collagen cross-linking regulator LOX. Inhibition of interstitial fibrosis by metformin is likely attributable to the activation of AMPK and the suppression of transforming growth factor-β1 (TGF-β1)/Smad3 signaling, leading to enhanced systemic insulin sensitivity. The ability of metformin to repress TGF-β1-induced fibrogenesis is abolished by the dominant negative AMPK in primary cells from the stromal vascular fraction. TGF-β1-induced insulin resistance is suppressed by AMPK agonists and the constitutively active AMPK in 3T3L1 adipocytes. In omental fat depots of obese humans, interstitial fibrosis is also associated with AMPK inactivation, TGF-β1/Smad3 induction, aberrant ECM production, myofibroblast activation, and adipocyte apoptosis. Collectively, integrated AMPK activation and TGF-β1/Smad3 inhibition may provide a potential therapeutic approach to maintain ECM flexibility and combat chronically uncontrolled adipose tissue expansion in obesity.

Matrix Metalloproteinase-9 Expression in Calcified Human Aortic Valves: A Histopathologic, Immunohistochemical, and Ultrastructural Study.

The hallmarks of calcific aortic valve disease (CAVD) are the significant quantitative and qualitative changes that occur in the extracellular matrix (ECM), which ultimately lead to increased leaflet stiffness and obstruction of left ventricular outflow. Mounting evidence suggests that ECM remodeling not only contribute to valve cell dysfunction but also alter certain cell signaling pathways responsible for the initiation and progression of the disease state. Matrix metalloproteinases (MMPs), collectively called matrixins, are a family of enzymes known to participate in numerous ECM remodeling events during embryonic development and in disease. The aim of the present study was to evaluate whether changes in MMP-9 expression might be involved in the pathophysiology of CAVD. For this purpose, we have analyzed a total of 19 pathologic valves from patients who underwent aortic valve replacement for calcific aortic stenosis. Microscopically, the cusp tissue showed diffuse fibrosis, neovascularization, and abnormal ECM remodeling with collagen disorganization and mineralization. Western blot and immunohistochemical analyses have been performed on both the areas overlying and remote from the mineral deposits. Protein expression data evidenced a significant upregulation of MMP-9 in the calcified lesion area. Consistent with these observations, immunohistochemistry demonstrated that MMP-9 protein was almost exclusively localized near or around the mineralized nodules, whereas was generally quite weak or absent in areas devoid of any calcification. Our data suggest that MMP-9 may play a key role in CAVD probably by promoting the fibrotic and procalcific remodeling of the ECM.

Gene polymorphism -418 G/C of tissue inhibitor of metalloproteinases 2 is associated with abdominal aortic aneurysm

The pathogenesis of abdominal aortic aneurysm (AAA) is connected with abnormal extracellular matrix remodeling, with the assistance of extracellular matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). A decrease in tissue inhibitor of metalloproteinases 2 (TIMP2) gene expression was detected in AAA patients. Recently, a -418 G/C (rs8179090) polymorphism of the TIMP2 gene promoter, influencing the transcription rate of the gene, has been described. This study investigated whether the -418 G/C gene polymorphism is associated with AAA in the Polish population. The TIMP2 gene promoter polymorphism was evaluated by polymerase chain reaction, followed by restriction enzyme analysis and pyrosequencing in 128 patients affected by AAA and in 180 individuals included as references. The control group was directly matched to patients according to known risk factors for vascular diseases. The frequency of the C allele was significantly higher in AAA patients than in the control group (odds ratio [OR], 2.516; P = .0005). The distribution of genotypes also differed significantly between the groups (CC + CG vs GG: OR, 2.906; P = .0037) or was close to being significantly different (CC vs GG + GC: OR, 2.144; P = .0501). A similar trend was observed in men but not in women. The multivariate logistic regression analysis indicated the TIMP2 gene promoter polymorphism is risk factor of AAA in the Polish population, with an adjusted OR of 4.99, when applied to a dominant inheritance model. Our study supports the hypothesis that TIMP2 and the -418G/C polymorphism located in the promoter of the TIMP2 gene are important in AAA pathophysiology.

Extracellular Matrix Remodeling: The Common Denominator in Connective Tissue DiseasesPossibilities for Evaluation and Current Understanding of the Matrix as More Than a Passive Architecture, but a Key Player in Tissue Failure

Increased attention is paid to the structural components of tissues. These components are mostly collagens and various proteoglycans. Emerging evidence suggests that altered components and noncoded modifications of the matrix may be both initiators and drivers of disease, exemplified by excessive tissue remodeling leading to tissue stiffness, as well as by changes in the signaling potential of both intact matrix and fragments thereof. Although tissue structure until recently was viewed as a simple architecture anchoring cells and proteins, this complex grid may contain essential information enabling the maintenance of the structure and normal functioning of tissue. The aims of this review are to (1) discuss the structural components of the matrix and the relevance of their mutations to the pathology of diseases such as fibrosis and cancer, (2) introduce the possibility that post-translational modifications (PTMs), such as protease cleavage, citrullination, cross-linking, nitrosylation, glycosylation, and isomerization, generated during pathology, may be unique, disease-specific biochemical markers, (3) list and review the range of simple enzyme-linked immunosorbent assays (ELISAs) that have been developed for assessing the extracellular matrix (ECM) and detecting abnormal ECM remodeling, and (4) discuss whether some PTMs are the cause or consequence of disease. New evidence clearly suggests that the ECM at some point in the pathogenesis becomes a driver of disease. These pathological modified ECM proteins may allow insights into complicated pathologies in which the end stage is excessive tissue remodeling, and provide unique and more pathology-specific biochemical markers.

Glomerular Expression of Hydrogen Peroxide-Inducible Clone-5 in Human and Rat Progressive Mesangial Proliferative Glomerulonephritis

Background/Aims: Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor-β₁ (TGF-β₁)- and hydrogen peroxide (H₂O₂)-inducible focal adhesion protein that may be necessary for maintaining the myofibroblastic phenotype in pathological scar formation. To investigate the involvement of Hic-5 in the pathogenesis of glomerulonephritis (GN), we examined the glomerular expression of Hic-5 in human and rat GN as well as the regulation of Hic-5 by TGF-β₁ in vitro. Methods and Results: Immunohistochemical analyses showed that the expression of Hic-5 was increased in mesangial cells (MCs) in human mesangial proliferative GN. Hic-5 expression was significantly correlated not only with the levels of α-smooth muscle actin (α-SMA) and TGF-β₁, the accumulation of extracellular matrix, and the number of glomerular cells, but also with the urinary protein level in patients with GN. Glomerular Hic-5 expression increased in parallel with α-SMA expression in a rat model of mesangial proliferative GN. Combined therapy with an angiotensin type I receptor blocker and an antioxidant in this model improved the histology and the expression of Hic-5 and α-SMA. TGF-β₁ upregulated Hic-5 and α-SMA protein levels in human cultured MCs. Conclusion: Our findings suggest that Hic-5 is involved in changes in the MC phenotype to produce abnormal extracellular matrix remodeling in GN.

Association of Metalloproteinase Gene Polymorphisms with Systemic Sclerosis in the European Caucasian Population

Systemic sclerosis (SSc) is classified among the complex genetic disorders and is characterized by massive extracellular matrix deposits. These may be due to overactivation of transforming growth factor ss that may be in part a result of abnormal remodeling of extracellular matrix and microfibrils. Metalloproteinases (MMP) are a family of proteolytic enzymes, and MMP 2, 9, and 14 contribute to the degradation of microfibrils. Our aim was to determine whether polymorphisms of the MMP2, MMP9, and MMP14 genes confer susceptibility to SSc in a large population. A case-control study was performed in 659 SSc patients and 511 healthy matched controls from a European Caucasian population. Six Tag single-nucleotide polymorphisms (SNP) of the MMP2 gene and 2 SNP of MMP9 and MMP14 genes were genotyped. All SNP were in Hardy-Weinberg equilibrium in the control population. There was no association between the MMP2, MMP9, and MMP14 variants we investigated and SSc for allelic and genotype frequencies. No association was observed for the different subphenotypes of SSc patients. Our results in a large cohort of European Caucasian SSc patients do not support that MMP2, MMP9, and MMP14 genes are involved in the genetic background of SSc.

Caveolin-1-dependent β1 integrin endocytosis is a critical regulator of fibronectin turnover

beta1 integrins are major cell surface receptors for fibronectin. Some integrins, including beta1 integrins, are known to undergo constitutive endocytosis and recycling. Integrin endocytosis/recycling has been implicated in the regulation of cell migration. However, the mechanisms by which integrin endocytosis/recycling regulates cell migration, and other biological consequences of integrin trafficking are not completely understood. We previously showed that turnover of extracellular matrix (ECM) fibronectin occurs via receptor-mediated endocytosis. Here, we investigate the biological relevance of beta1 integrin endocytosis to fibronectin matrix turnover. First, we demonstrate that beta1 integrins, including alpha5beta1 play an important role in endocytosis and turnover of matrix fibronectin. Second, we show that caveolin-1 constitutively regulates endocytosis of alpha5beta1 integrins, and that alpha5beta1 integrin endocytosis can occur in the absence of fibronectin and fibronectin matrix. We also show that downregulation of caveolin-1 expression by siRNA results in marked reduction of beta1 integrin and fibronectin endocytosis. Hence, caveolin-1-dependent beta1 integrin and fibronectin endocytosis plays a critical role in fibronectin matrix turnover, and may contribute to abnormal ECM remodeling that occurs in fibrotic disorders.

Expression of matrix metalloproteinases MMP-2 and MMP-9 is altered during nephrogenesis in fetuses from diabetic rats

Remodeling of extracellular matrix (ECM) is an important physiological feature of normal growth and development. Recent studies have emphasized the role of matrix metalloproteinases (MMP-2 and MMP-9) in normal mouse nephrogenesis. We have demonstrated previously in the rat that in utero exposure to maternal diabetes impairs renal development leading to a 30% reduction in the nephron number. Transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) are known to mediate high glucose effects on matrix degradation. The aim of the present study was to address the expression of type IV collagenase and TGF-β1/CTGF systems in rat kidney during normal development and after in utero exposure to maternal diabetes. Both MMP-2 and MMP-9 mRNA metanephric expressions and activities were dramatically downregulated in kidneys issued from diabetic fetuses and in metanephros cultured in the presence of high glucose concentration. TGF-β1 and CTGF expressions were significantly enhanced in diabetic fetal kidneys and in high glucose cultured metanephroi. Conditioned media obtained from metanephroi grown with high glucose concentration upregulated functional TGF-β activity in transfected ATDC5 cells. In conclusion, in impaired nephrogenesis resulting from in utero exposure to maternal diabetes, alteration of both type IV collagenase and TGF-β1/CTGF systems may lead to abnormal remodeling of ECM, which may, in turn, induce defects in ureteral bud branching leading to the observed reduction in the nephron number with consequences later in life: progression of chronic renal disease and hypertension.

Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix.

To investigate the effect of IOP on retinal ganglion cell (RGC) apoptosis and correlate the effects with IOP-induced changes in extracellular matrix (ECM) in the retina and optic nerve head (ONH) in glaucomatous rat eyes. Thirty-seven Dark Agouti rats had elevated IOP induced in the left eye by hypertonic saline episcleral vein injections. Eyes were examined at 3 months histologically for RGC apoptosis and expression of specific ECM components. RGC apoptosis was significantly related to IOP exposure (integral DeltaIOP P <0.001; peak IOP P <0.01). In the RGC layer, elevated IOP correlated positively to a significant increase in MMP-9 activity (P <0.001), tissue inhibitor of matrix metalloproteinase (TIMP-1) (P <0.05), and collagen I (P <0.01), and negatively correlated to deposition of laminin (P <0.05) and TGF-beta2 (P <0.05). There was a significant correlation between MMP-9 activity and both RGC apoptosis (P <0.001) and loss of laminin (P <0.01). IOP exposure was also associated with increased deposition of TGF-beta2 and collagen I at the ONH (P <0.01). The results demonstrated that RGC apoptosis in glaucoma correlates strongly with elevated IOP and is significantly associated with IOP-induced changes in specific ECM components in the RGC layer. The study shows for the first time a link between MMP-9, laminin degradation, RGC apoptosis, and IOP exposure in glaucoma. The findings suggest that abnormal ECM remodeling in the glaucomatous retina may relate to RGC death and support the notion that the retina is a primary site of injury in glaucoma.