Dessislava Markova

A comparative analysis of the fibulin protein family. Biochemical characterization, binding interactions, and tissue localization.

Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of ∼20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins.

Genetic Heterogeneity of Cutis Laxa: A Heterozygous Tandem Duplication within the Fibulin-5 (FBLN5) Gene

Inherited cutis laxa is a connective tissue disorder characterized by loose skin and variable internal organ involvement, resulting from paucity of elastic fibers. Elsewhere, frameshift mutations in the elastin gene have been reported in three families with autosomal dominant inheritance, and a family with autosomal recessive cutis laxa was recently reported to have a homozygous missense mutation in the fibulin-5 gene. In the present study, we analyzed the gene expression of elastin and fibulins 1-5 in fibroblasts from five patients with cutis laxa. One patient was found to express both normal (2.2 kb) and mutant (2.7 kb) fibulin-5 mRNA transcripts. The larger transcript contains an internal duplication of 483 nucleotides, which resulted in the synthesis and secretion of a mutant fibulin-5 protein with four additional tandem calcium-binding epidermal growth factor-like motifs. The mutation arose from a 22-kb tandem gene duplication, encompassing the sequence from intron 4 to exon 9. No fibulin-5 or elastin mutations were detected in the other patients. The results demonstrate that a heterozygous mutation in fibulin-5 can cause cutis laxa and also suggest that fibulin-5 and elastin gene mutations are not the exclusive cause of the disease.

TNF-α and IL-1β Promote a Disintegrin-like and Metalloprotease with Thrombospondin Type I Motif-5-mediated Aggrecan Degradation through Syndecan-4 in Intervertebral Disc

Background: Disc degeneration is characterized by elevated levels of cytokines, TNF-α and IL-1β and an increase in aggrecan-rich matrix degradation. Results: Cytokine-dependent syndecan-4 expression regulate ADAMTS-5/aggrecanse2 activation in disc cells. Conclusion: Syndecan-4 may play a key role in pathogenesis of degenerative disc disease. Significance: Syndecan-4 may offer a potential therapeutic target for controlling progression of degenerative disc disease. Elevated levels of TNF-α, IL-1β and a resultant increase in ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type I motifs) expression is seen during disc degeneration. However, if these pro-inflammatory cytokines control ADAMTS activity is not definitively known. The goal of the investigation was to study if TNF-α and IL-1β regulate syndecan-4 (SDC4) expression, and if SDC4 was responsible for promoting aggrecan degradation through controlling ADAMTS activity in nucleus pulposus cells of the intervertebral disc. Cytokine treatment increased SDC4 expression and promoter activity. Use of inhibitor, SM7368 and co-transfections with IκBα, RelA/p50 showed that NF-κΒ regulated both basal and cytokine-dependent SDC4 transcription. SDC4 promoter harboring RelA binding site mutation was unresponsive to the cytokines. Moreover, cytokines failed to increase SDC4 promoter activity in RelA-null cells. Cytokines increased ADAMTS-4/5 expression and aggrecan degradation and promoted SDC4 interaction with ADAMTS-5. Treatment with heparinase-III and p-nitrophenyl-β-d-xylopyranoside (PNPX), an inhibitor of heparan sulfate synthesis and transfection with SDC4-shRNA partially blocked cytokine mediated aggrecan degradation. Analysis of human tissues showed increased aggrecan degradation with a concomitant increase in SDC4 and ADAMTS-5 protein expression with severity of disc disease. Likewise, SDC4, TNF-α, IL-1β, ADAMTS-4, and ADAMTS-5 mRNA expression increased in degenerate tissues. We conclude that in nucleus pulposus, TNF-α and IL-1β regulate SDC4 expression, which plays a key role in pathogenesis of degenerative disc disease by promoting aggrecan degradation by ADAMTS-5.

Compound heterozygous mutations in fibulin-4 causing neonatal lethal pulmonary artery occlusion, aortic aneurysm, arachnodactyly, and mild cutis laxa†‡

Mutations involving elastic tissue proteins result in a broad spectrum of phenotypes affecting skin, skeleton, ocular and vascular structures, including tortuous blood vessels and cutis laxa. Here we report on a female newborn with apparently long fingers, aortic aneurysm, tortuous pulmonary arteries and mild generalized lax skin. She died at 27 days of age due to severe respiratory distress and inoperable systemic vascular abnormalities. Skin biopsy showed marked paucity and fragmentation of elastic fibers and autopsy revealed occlusion of the pulmonary artery. DNA analysis identified compound heterozygous mutations ((c.835C > T (p.R279C)/c.1070_1073dupCCGC) in fibulin‐4, a recently recognized elastic fiber associated protein. Analyses of dermal fibroblasts from the patient indicated that fibulin‐4 mRNAs with the 4‐bp duplication transcribed from one allele are probably subject to nonsense‐mediated decay, whereas synthesis and secretion of the missense R279C fibulin‐4 protein from the other allele is severely impaired. Immunostaining demonstrated a total absence of fibulin‐4 fibers in the extracellular matrix deposited by the patients fibroblasts. Our studies provide evidence that deficiency in fibulin‐4 leads to a perinatal lethal condition associated with elastic tissue abnormalities.

Expression and relationship of proinflammatory chemokine RANTES/CCL5 and cytokine IL-1β in painful human intervertebral discs.

Study Design. Laboratory study. Objective. To evaluate expression of chemokine regulated and normal T cell expressed and secreted (RANTES)/C-C motif ligand 5 (CCL5) and interleukins in intervertebral discs (IVDs) specimens from patients with discogram-proven painful degeneration. Summary of Background Data. Discogenic back pain results in tremendous costs related to treatment and lost productivity. The relationship between inflammation, degeneration (IVD), and cytokine upregulation is well established, but other mediators of the inflammatory cascade are not well characterized. Methods. Painful IVDs were taken from 18 patients undergoing surgery for discogenic pain with positive preoperative discogram. Painless control tissue was taken at autopsy from patients without back pain/spinal pathology or spinal levels with negative discograms resected for deformity. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to evaluate RANTES, IL-1&bgr;, IL-6, and IL-8 expression in painful and control discs. RANTES and interleukin expression were analyzed on the basis of Pfirrmann grade. Disc cells were cultured in alginate beads using 2 groups: an untreated group and a group treated with 10 ng/mL IL-1&bgr;, 10 ng/mL TNF-&agr;, and 1% fetal bovine serum to induce a degenerative phenotype. Results. Nine painless IVD specimens and 7 painful IVD specimens were collected. RANTES expression demonstrated a 3.60-fold increase in painful discs versus painless discs, a significant difference (P = 0.049). IL-1&bgr; expression demonstrated significantly higher expression in painful discs (P = 0.03). RANTES expression data demonstrated significant upregulation with increasing Pfirrmann grade (P = 0.045). RANTES expression correlated significantly with IL-1&bgr; expression (&rgr; = 0.67, P < 0.0001). RANTES expression increased more than 200-fold in the alginate culture model in cells treated with IL-1&bgr;/TNF-&agr;, 1% fetal bovine serum (P < 0.001). Conclusion. RANTES and IL-1&bgr; expression was significantly elevated in painful IVDs after careful selection of painless versus painful IVD tissue. RANTES expression was found to correlate significantly with expression of IL-1&bgr;. RANTES was upregulated by IL-1&bgr;/TNF-&agr;/1% fetal bovine serum an in vitro treatment to induce a degenerative phenotype.

Regulation of CCN2/connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: role of Smad and activator protein 1 signaling.

OBJECTIVE To investigate transforming growth factor beta (TGFbeta) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. METHODS Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFbeta-mediated CTGF promoter activity. RESULTS CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGFbeta treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFbeta-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFbeta was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGFbeta expression in the degenerated state. CONCLUSION TGFbeta, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Post-ischemic myocardial fibrosis occurs independent of hemodynamic changes.

OBJECTIVES Myocardial fibrosis is a major component of ventricular remodeling after large myocardial infarction (MI). The present study tests the hypothesis that post-ischemic myocardial fibrosis can occur independent of hemodynamic changes. METHODS A mouse model of distal left coronary artery ligation was established to induce a small infarct (less than 15% of the left ventricle) in order to avoid significant mechanical overload after permanent myocardial ischemia. Left heart catheterization was performed to evaluate the post-infarct hemodynamics. Tissues from both ischemic and non-ischemic myocardium were examined for mRNA and protein expression at 24, 72 h and 7 days after ligation. RESULTS Heart/body weight ratio after ligation was increased by approximately 10% over sham control although there is no statistically significant difference in hemodynamic parameters between the two groups. Non-ischemic myocardium distant from the infarct site showed molecular evidence of myocardial fibrosis 72 h and 7 days after ligation. There was marked up-regulation of mRNAs for extracellular matrix (ECM) proteins and their cross-linking enzyme, such as collagens type I, III and VI, and lysyl oxidase. Immunohistochemical study confirmed that the expression of these ECM proteins was significantly increased in the non-ischemic myocardium after 7 days. TGF-beta1 was up-regulated after 72 h in both ischemic and non-ischemic myocardium. CONCLUSIONS Molecular and histopathological findings demonstrate that abnormal myocardial fibrosis can be induced by a small infarct independent of secondary hemodynamic changes.

Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Intervertebral Disc DISTINCT ROLES OF PHD2 AND PHD3 PROTEINS IN CONTROLLING HIF-1α ACTIVITY IN HYPOXIA

Background: Regulation of PHD expression and their function in hypoxia is unknown in nucleus pulposus. Results: Expression of PHD1–3 is regulated differentially by HIF-1/2. Under hypoxia, PHD2 degrades HIF-1α, whereas PHD3 promotes its activity. Conclusion: In hypoxic nucleus pulposus, HIFs and PHDs constitute a regulatory network. Significance: PHD2 and 3 may serve as oxygen sensors in the intervertebral disc. Adaptive response to hypoxia in nucleus pulposus cells of the intervertebral disc is regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Moreover, oxygen-dependent turnover of HIF-1α in these cells is controlled by the prolyl-4-hydroxylase domain (PHD) family of proteins. Whether HIF homologues control expression of PHDs and whether PHDs control hypoxia-inducible factor (HIF) turnover and/or activity under hypoxia is not known. Here, we show that in nucleus pulposus cells, hypoxia robustly induces PHD3 expression and, to a lesser extent, of PHD2 and PHD1. Reporter analysis shows that the hypoxic induction of the PHD2 promoter is HIF-1α dependent, whereas PHD3 promoter/enhancer activity is dependent on both HIF-1α and HIF-2α. Lentiviral delivery of HIF-1α, ShHIF-1α, and ShHIF-1β confirmed these observations. Noteworthy, HIF-1α maintains basal expression of PHD1 in hypoxia at the posttranscriptional level. Finally, loss of function studies using lentiviral transduction of ShPHDs clearly shows that even at 1% O2, PHD2 selectively degrades HIF-1α. In contrast, in hypoxia, PHD3 enhances HIF-1α transcriptional activity without affecting protein levels. To correlate these observations with disc disease, a condition characterized by tissue vascularization, we analyzed human tissues. Increased PHD1 mRNA expression but decreased PHD2 and PHD3 expression is observed in degenerate tissues. Interestingly, the hypoxic responsiveness of all the PHDs is maintained in isolated nucleus pulposus cells regardless of the disease state. We propose that PHD2 and PHD3 can be used as a biomarker of tissue oxygenation in the disc and that, as such, it may have important clinical implications.

Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

ABSTRACT Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.

Histological Features of the Degenerating Intervertebral Disc in a Goat Disc-injury Model

Study Design. An in vivo study to develop a goat large-animal model for intervertebral disc (IVD) degeneration. Objective. To determine an optimal method for inducing goat IVD degeneration suitable for testing disc regeneration therapies. Summary of Background Data. Although rodent, rabbit, and other small animal studies are useful, the narrow dimensions of IVDs in these species limit studies requiring injection of a relevant volume of therapeutics or implantation of engineered tissue constructs. For this study, the goat was selected because the size and shape of their IVDs are comparable with those of adult humans. Methods. A minimally invasive approach that did not cause significant morbidity or mortality to adult goats (n = 6) was used. Under fluoroscopic guidance, goat lumbar IVDs were injured with a 4.5-mm drill bit or #15 or #10 surgical blades. Two months postinjury, the goats were killed and their IVDs with adjacent end plates were isolated, decalcified, and stained. Results. A numerical histologic scale to categorize the degree of goat IVD degeneration was developed on the basis of the histologic features of rabbit IVDs previously described by Masuda et al, goat IVDs described by Hoogendoorn et al, and human IVDs described by Boos et al. The interrater agreement of our scoring system was assessed (weighted kappa value = 0.6646). Mann-Whitney U tests were used to compare the injured IVDs with uninjured control. A 4.5-mm drill bit inserted to a 15-mm depth resulted in a significantly higher histologic score than uninjured controls (P = 0.01). Injury with a #15 or #10 blade did not result in increased histologic scores compared with uninjured controls. Conclusion. A comparison of the various injuries inflicted showed that the use of a 4.5-mm drill bit resulted in the most significant histologic changes.