Site-1 and site-2 proteases: A team of two in regulated proteolysis.

The site-1 and site-2 proteases (S1P and S2P) were identified over 20 years ago, and the functions of both have been addressed in numerous studies ever since. Whereas S1P processes a set of substrates independently of S2P, the latter acts in concert with S1P in a mechanism, called regulated intramembrane proteolysis, that controls lipid metabolism and response to unfolded proteins. This review summarizes the molecular roles that S1P and S2P jointly play in these processes. As S1P and S2P deficiencies mainly affect connective tissues, yet with varying phenotypes, we discuss the segregated functions of S1P and S2P in terms of cell homeostasis and maintenance of the connective tissues. In addition, we provide experimental data that point at S2P, but not S1P, as a critical regulator of cell adaptation to proteotoxicity or lipid imbalance. Therefore, we hypothesize that S2P can also function independently of S1P activity.

Immunolocalization of aromatase P450 in the epididymis of Podarcis sicula and Rattus rattus.

The goal of this study was to evaluate P450 aromatase localization in the epididymis of two different vertebrates: the lizard Podarcis sicula, a seasonal breeder, and Rattus rattus, a continuous breeder. P450 aromatase is a key enzyme involved in the local control of spermatogenesis and steroidogenesis and we proved for the first time that this enzyme is represented in the epididymis of both P. sicula and R. rattus. In details, P450 aromatase was well represented in epithelial and myoid cells and in the connective tissue of P. sicula epididymis during the reproductive period; instead, during autumnal resumption this enzyme was absent in the connective tissue. During the non-reproductive period, P450 aromatase was localized only in myoid cells of P. sicula epididymis, whereas in R. rattus it was localized both in myoid cells and connective tissue. Our findings, the first on the epididymis aromatase localization in the vertebrates, suggest a possible role of P450 aromatase in the control of male genital tract function, particularly in sperm maturation.

The enzyme profiles in the connective tissue attaching pin bones to the surrounding tissue is specific in farmed salmon (Salmo salar) and cod (Gadus morhua L.).

Post mortem storage is a necessary process for removal of pin bones without destruction of fillets, thereby avoiding volume and economic loss. However, the enzymes involved in loosening pin bones during storage have not been studied to a great extent. In this study, the activities and localization of MMPs in the connective tissue (CT) of pin bones dissected from fillet of salmon and cod were investigated. Interestingly, the enzyme activity profile in these two species was different during post mortem storage of fish fillets. Adding MMP inhibitor (GM6001) and serine protease inhibitor (Pefabloc) revealed different effects in the two species, suggesting different regulations in salmon and cod. In situ zymography with the same inhibitors verified MMP and serine protease activity in CT close to pin bone at early post mortem (6 h) in salmon. However, MMP inhibition was not evident in cod in this area at that time point. Immunohistochemistry further revealed MMP9 and MMP13 were located more to the outer rim of CT, facing the pin bone and adipose tissue, while MMP7 was more randomly distributed within CT in salmon. In contrast, all these three MMPs were randomly distributed in CT in cod. In summary, our study reveals different MMP enzyme profiles in salmon and cod in the pin bone area, influenced by serine proteases, and suggests that MMPs and serine proteases must be taken in consideration when studying the conditions for early pin bone removal.

Variable patterns of ectopic mineralization in Enpp1asj-2J mice, a model for generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder characterized by early onset of extensive mineralization of the cardiovascular system. The classical forms of GACI are caused by mutations in the ENPP1 gene, encoding a membrane-bound pyrophosphatase/phosphodiesterase that hydrolyzes ATP to AMP and inorganic pyrophosphate. The asj-2J mouse harboring a spontaneous mutation in the Enpp1 gene has been characterized as a model for GACI. These mutant mice develop ectopic mineralization in skin and vascular connective tissues as well as in cartilage and collagen-rich tendons and ligaments. This study examined in detail the temporal ectopic mineralization phenotype of connective tissues in this mouse model, utilizing a novel cryo-histological method that does not require decalcification of bones. The wild type, heterozygous, and homozygous mice were administered fluorescent mineralization labels at 4 weeks (calcein), 10 weeks (alizarin complexone), and 11 weeks of age (demeclocycline). Twenty-four hours later, outer ears, muzzle skin, trachea, aorta, shoulders, and vertebrae were collected from these mice and examined for progression of mineralization. The results revealed differential timeline for disease initiation and progression in various tissues of this mouse model. It also highlights the advantages of cryo-histological fluorescent imaging technique to study mineral deposition in mouse models of ectopic mineralization disorders.

Immunohistochemical expression of matrix metalloproteinases in ameloblastomas and pericoronal follicles.

BACKGROUND: Ameloblastoma (AM) is a benign odontogenic neoplasm characterized by local invasiveness and recurrence. We compared the immunohistochemical expression of matrix metalloproteinases in different clinical types of AM as well as in normal odontogenic tissue. METHODS: Thirteen cases of solid AMs, five cases of unicystic AM and eight pericoronal follicles (PF) were selected and subjected to immunohistochemical investigation for matrix metalloproteinase-1, matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions. RESULTS: The expressions of MMP-1 and MMP-2 were very high in the cytoplasm of cells throughout the entire epithelium and in fibroblasts from the adjacent connective tissue. MMP-9 expression was observed in the same location although with weaker staining. The Kruskal-Wallis test showed statistically significant differences in the epithelial expressions of MMP-1 and MMP-2; there was lower expression among solid AMs when compared with unicystic AM and PF. Compared to both types of AM, higher stromal expression of MMP-9 was found in PF. CONCLUSION: MMP-1, MMP-2 and MMP-9 seem to be associated with AM tumour behaviour as well as physiological tissue remodelling within PF.

Telomerase expression in individuals with chronic and aggressive periodontitis.

BACKGROUND: The aim of this study is to evaluate the expression of human telomerase reverse transcription (hTERT) enzyme in chronic periodontitis (CP) and aggressive periodontitis (AgP) compared with healthy individuals. METHODS: A total of 79 individuals consented to participate in the study. The study sample comprised healthy individuals (n = 30), patients with CP (n = 30), and patients with AgP (n = 19). Gingival tissue was collected and evaluated for hTERT expression by Western blot and immunohistochemical methods. Reverse transcription polymerase chain reaction was performed using the gingival crevicular fluid (GCF) samples. RESULTS: The hTERT messenger RNA (mRNA) and protein expression was significantly higher in AgP compared with CP (P <0.001). In GCF, 53.33% of patients with CP and 68.42% of patients with AgP were showing hTERT mRNA expression, but it was not detected in the control group. The AgP tissue showed higher hTERT expression compared with CP (P <0.001). The hTERT mRNA expression did not show a correlation with gingival index (GI), plaque index (PI), probing depth (PD), and clinical attachment loss (AL) in patients with AgP, whereas hTERT protein expression was strongly correlated with GI, PI, PD, and AL in patients with AgP. The protein expression of hTERT shows significant but moderate correlation with GI and AL in patients with CP. CONCLUSION: High expression of hTERT might be associated with periodontal disease progression, suggesting that hTERT could be a potential prognostic marker.

Matrix metalloproteinase expression in keratocystic odontogenic tumors and primary cells.

UNLABELLED: Keratocystic odontogenic tumors (KCOTs) are locally invasive, rapidly proliferating cystic lesions of the jaw. The bone-invasive nature of these tumors has been previously associated with the expression of matrix metalloproteinases (MMPs), which degrade the extracellular matrix. The purpose of this study was to assess the expression and activity of MMPs in primary KCOT cells and tumor tissue. METHODS: Four independently established KCOT primary cell populations were grown in Dulbecco's modified Eagle medium supplemented with 10% FBS and antibiotics. Primary cells were analyzed by qRT-PCR and immunohistochemistry (IHC), and for secretion of active MMPs. Primary tumor sections were analyzed by IHC. RESULTS: Of the 18 human MMPs examined, 9 were consistently expressed in primary KCOT cells. MMP-2 and MMP-14 were highly expressed in all KCOT populations, while MMP-1, 3, 11, 12, 16, 17, and 19 were moderately expressed. MMP-3, 11, 12, 16, 17 and 19 were shown to be expressed in KCOTs for the first time. No significant differences in MMPS profiles were found between syndromic (KCOT-3) and non-syndromic cell populations (KCOT-1/2/4). Protein expression of MMP-1, 11, 12, 14 and 16 was confirmed in each KCOT cell populations by IHC. KCOT-3 cells secreted active MMP-2 as determined by a gel zymography assay. Expression of MMP-1, 2, 3, 11, 12, 14, and 16 was confirmed in matching primary KCOT tumor sections representing syndromic and non-syndromic KCOTs. CONCLUSION: KCOT primary cell populations and tumors express a wide range of MMPs, which likely play a role in the bone-invasive nature of these tumors.

Role of PTPα in the destruction of periodontal connective tissues.

IL-1ß contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα(+/+) and PTPα(-/-) mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1ß. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5-3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα(-/-) mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1ß or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1ß stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα(KO) and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1ß. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1ß-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1ß signaling through focal adhesions.

Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA.

The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.

The roles of matrix metalloproteinases-2, -7, -10 and tissue inhibitor of metalloproteinase-1 in the pathogenesis of oral lichen planus.

BACKGROUND: The aim of this study was to investigate the roles of Matrix Metalloproteinases (MMP)-2, Metalloproteinases-7, Metalloproteinases-10 and Tissue Inhibitors of Metalloproteinase-1 (TIMP-1) in the pathogenesis of oral lichen planus (OLP) disease in same tissue samples. METHODS: Thirty-nine individuals [29 patients with OLP (74%) and 10 healthy control subjects (25%)] were included in our study. The mean age was 48 ± 14.39 with a range of 20-75. RESULTS: MMP-2 and MMP-7 expression was significantly different in the patient and control groups in the epithelium and the connective tissue (P<0. 05). The ratio of MMP-2/TIMP-1 and MMP-7/TIMP-1 were higher in patient with OLP group than control group. CONCLUSIONS: Along with the exposure of the role of MMPs activity on diseases characterized by the tissue destruction, several studies were conducted on the pharmacological control of MMPs activity. However, understanding of the biological functions of MMPs is very important for the development and implementation of MMP inhibitors in the treatment of diseases. According to the results of this study, we suggest that MMP-2, MMP-7, and TIMP-1 may be involved in the formation of OLP lesions. Further studies on MMPs may be useful for understanding and treating the diseases such as OLP.

Co-expression of endothelial and neuronal nitric oxide synthases in the developing vasculatures of the human fetal eye.

BACKGROUND: Nitric oxide (NO) is a multifunctional gaseous molecule that regulates various physiological functions in both neuronal and non-neuronal cells. NO is synthesized by nitric oxide synthases (NOSs), of which three isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) constitutively produce low levels of NO as a cell-signaling molecule in response to an increase in intracellular calcium concentration. Recent data have revealed a predominant role of eNOS in both angiogenesis and vasculogenesis. METHODS: The immunohistochemical localization of nNOS and eNOS was investigated during embryonic and fetal ocular vascular development from 7 to 21 weeks gestation (WG) on sections of cryopreserved tissue. RESULTS: eNOS was confined to endothelial cells of developing vessels at all ages studied. nNOS was prominent in nuclei of vascular endothelial and smooth muscle cells in the fetal vasculature of vitreous and choriocapillaris. nNOS was also prominent in the nuclei of CXCR4(+) progenitors in the inner retina and inner neuroblastic layer. CONCLUSIONS: These findings demonstrate co-expression of n- and eNOS isoforms in different compartments of vasoformative cells during development. Nuclear nNOS was present in vascular and nonvascular progenitors as well as endothelial cells and pericytes. This suggests that nNOS may play a role in the transcription regulatory systems in endothelial cells and pericytes during ocular hemo-vasculogenesis, vasculogenesis, and angiogenesis.

Protein kinase C-delta mediates adventitial cell migration through regulation of monocyte chemoattractant protein-1 expression in a rat angioplasty model.

OBJECTIVE: The adventitia is increasingly recognized as an important player during the development of intimal hyperplasia. However, the mechanism of adventitial cell recruitment to the subintimal space remains largely undefined. We have shown previously that gene transfer of protein kinase C-delta (PKCδ) increases apoptosis of smooth muscle cells following balloon injury. In the current study, we investigated a potential role of PKCδ in regulating the recruitment of adventitial cells. METHODS AND RESULTS: Conditioned media from PKCδ-overexpressing smooth muscle cells stimulated migration and CCR2 expression of adventitial fibroblasts through a MCP-1 dependent mechanism. Following balloon injury of rat carotid arteries, overexpression of PKCδ in smooth muscle cells significantly increased MCP-1 and CCR2 expression and the number of adventitia-originated cells detected in the neointima. Administration of an anti-MCP-1 antibody markedly diminished the recruitment of adventitial cells. Combined PKCδ overexpression and anti-MCP-1 inhibited intimal hyperplasia more effectively than either approach alone. CONCLUSIONS: Our data suggest that PKCδ regulates recruitment of adventitial cells to the neointima via a mechanism involving upregulation of the MCP-1/CCR2 signaling axis in injured arteries. Blockage of MCP-1 while enhancing apoptosis may serve as a potential therapeutic strategy to attenuate intimal hyperplasia.

Cyclooxygenase-2 expression in gingival biopsies from periodontal patients is correlated with connective tissue loss.

BACKGROUND: The objective of this study is to compare cyclooxygenase-2 (COX-2) protein expression in gingival biopsies from patients with chronic periodontitis (CP), patients with gingivitis (GV), and individuals with no periodontal disease (control group) and to establish its relationship with clinical variables and connective tissue loss in the lamina propria. METHODS: A cross-sectional and analytic study was conducted in 108 gingival biopsies from 52 patients with CP, 39 with GV, and 17 controls. All biopsies were processed for conventional histopathologic study, immunohistochemical determination of COX-2 protein expression, and automatic quantification of connective tissue by image analysis. RESULTS: The protein expression of COX-2, mainly produced by plasma cells and monocytes, was significantly related to the presence of periodontal disease, bleeding index, intensity of inflammatory infiltrate, and loss of connective tissue in the lamina propria of gingival biopsies (P <0.01, Spearman test). COX-2 expression was also directly correlated with attachment loss (P <0.05, Spearman test). CONCLUSIONS: COX-2 protein expression is higher in patients with GV and CP than in individuals without periodontal disease and is inversely correlated with the amount of connective tissue in the lamina propria as determined by image analysis. This finding suggests that COX-2 participates in mechanisms and pathway signaling related to the destruction of fibrillar support structures of the periodontium.

Matrix metalloproteinase-1 of gingival fibroblasts influenced by advanced glycation end products (AGEs) and their association with receptor for AGEs and nuclear factor-κB in gingival connective tissue.

BACKGROUND: The effect of advanced glycation end products (AGEs) on gingival inflammation has not been fully elucidated. This study aims to investigate the hypothesis that AGEs may enhance the expression of matrix metalloproteinase-1 (MMP-1) of human gingival fibroblasts (HGFs) and to explore whether the signal pathway receptor for AGE (RAGE)/nuclear factor-κB (NF-κB) are involved in the expression of MMP-1 in HGFs. METHODS: Cultured HGFs from 12 healthy gingival human tissue samples were coincubated with AGEs for the detection of MMP-1 protein and mRNA. Thirty-six gingival samples were collected and treated for the determination of RAGE, NF-κB, and MMP-1 mRNA level in gingival connective tissues from the participants with chronic periodontitis, diabetes-associated periodontitis, and healthy controls. Enzyme-linked immunosorbent assay and real-time fluorescence reverse transcription-polymerase chain reaction were used for the measurement of protein and mRNA level, respectively. In addition, clinical periodontal parameters were also checked. RESULTS: AGEs strongly induced MMP-1 mRNA and protein expression in HGFs and in a time- and concentration-dependent manner (P <0.05). In gingival connective tissue, the level of both RAGE mRNA and NF-κB mRNA were higher in patients with periodontitis than in healthy controls (P <0.05). There was significant correlation between the level of RAGE mRNA and NF-κB mRNA (R(2) = 0.90, P <0.05). CONCLUSIONS: Accumulation of AGEs may upregulate the expression of MMP-1 by HGFs, which may play a role in the development of diabetes-associated periodontitis, and RAGE/NF-κB pathway may be involved in metabolism of MMP-1 in HGFs.

Excessive fluoride intake alters the MMP-2, TIMP-1 and TGF-ß levels of periodontal soft tissues: an experimental study in rabbits.

OBJECTIVES: This study evaluated the influence of fluoride on periodontal soft tissues by investigating any alterations in their MMP-2, TIMP-1 and TGF-ß profiles secondary to excessive fluoride intake. MATERIAL AND METHODS: Fluorosis was induced in 18 rabbits (test group) through consumption of fluoride added to drinking water, whereas 10 rabbits consumed regular tap water as daily supply (control group). Following fluorosis verification, animals were sacrificed and their 1st mandibular molar teeth were utilized in the assessments. MMP-2, TIMP-1 and TGF-ß were separately investigated for gingival epithelium (GE), gingival connective tissue (GC) and periodontal ligament (PL) to evaluate periodontal soft tissues. Histological sections were prepared from the groups, the parameters were determined by immunohistochemistry, and their levels were calculated by quantification of the immunostainings. RESULTS: Staining intensity of MMP-2 in GC and PL (p < 0.01); TIMP-1 and TGF-ß of GE, GC and PL (p < 0.01) were higher in the test group compared to those of the control group. Intra-group staining of TIMP-1 was higher than MMP-2 in all test group compartments (p < 0.01) and in the control group GE (p < 0.01). TIMP-1 was also higher than TGF-ß in the GE and PL of the test group (p < 0.05) and in the GE of the control group (p < 0.01). CONCLUSION: These results suggest that excessive fluoride intake may affect periodontal soft tissues by increasing MMP-2, TIMP-1 and TGF-ß, and thereby altering the MMP-2/TIMP-1 and TIMP-1/TGF-ß ratios. CLINICAL RELEVANCE: Excessive fluoride consumption may alter the periodontal tissue homeostasis which may be detrimental in the maintenance of periodontal health.

Comparative analysis of the immunohistochemical expression of collagen IV, MMP-9, and TIMP-2 in odontogenic cysts and tumors.

OBJECTIVE: The aim of this study was to evaluate the immunohistochemical expression of collagen IV, matrix metalloproteinase (MMP) 9 and tissue inhibitor of MMP (TIMP) 2 in dentigerous cysts (DCs), radicular cysts (RCs), keratocystic odontogenic tumors (KOTs), and ameloblastomas. STUDY DESIGN: Twenty cases of DCs, 20 RCs, 20 KOTs, and 20 ameloblastomas were selected and analyzed by immunohistochemistry. RESULTS: Most DCs and RCs showed continuous and >50% staining for collagen IV in the basement membrane of the epithelium, whereas predominantly discontinuous thin and ≤ 50% staining was observed in KOTs and ameloblastomas, with a significant difference in staining percentage (P < .001). MMP-9 was diffusely distributed and localized in both epithelial and mesenchymal cells of all of the lesions analyzed. The staining percentage was higher in the epithelium (P = .058) and mesenchyme (P = .005) of KOTs and ameloblastomas. Moreover, the distribution pattern, location, and percentage of expression of TIMP-2 were similar in the lesions studied, except for ameloblastoma, with a significant difference in staining percentage (P < .001). CONCLUSION: These results demonstrate that the interaction between collagen IV, MMP-9, and TIMP-2 is an important factor for the establishment of differences in the biologic behavior of the odontogenic cysts and tumors studied.

Dermatitis herpetiformis sera or goat anti-transglutaminase-3 transferred to human skin-grafted mice mimics dermatitis herpetiformis immunopathology.

Dermatitis herpetiformis (DH) is characterized by deposition of IgA in the papillary dermis. However, indirect immunofluorescence is routinely negative, raising the question of the mechanism of formation of these immune deposits. Sárdy et al. (2002. J. Exp. Med. 195: 747-757) reported that transglutaminase-3 (TG3) colocalizes with the IgA. We sought to create such deposits using passive transfer of Ab to SCID mice bearing human skin grafts. IgG fraction of goat anti-TG3 or control IgG were administered i.p. to 20 mice. Separately, sera from seven DH patients and seven controls were injected intradermally. Biopsies were removed and processed for routine histology as well as direct immunofluorescence. All mice that received goat anti-TG3 produced papillary dermal immune deposits, and these deposits reacted with both rabbit anti-TG3 and DH patient sera. Three DH sera high in IgA anti-TG3 also produced deposits of granular IgA and TG3. We hypothesize that the IgA class anti-TG3 Abs are directly responsible for the immune deposits and that the TG3 is from human epidermis, as this is its only source in our model. These deposits seem to form over weeks in a process similar to an Ouchterlony immunodiffusion precipitate. This process of deposition explains the negative indirect immunofluorescence results with DH serum.

Inhibition of inducible nitric oxide synthase promotes vein graft neoadventitial inflammation and remodelling.

BACKGROUND: Grafting veins into the arterial circulation causes endothelial damage and neointimal hyperplasia. However, the remodelling of vein grafts and the contribution of the endothelium is not well understood. Since nitric oxide (NO) has a crucial role in vascular function, we investigated the importance of NO synthases (NOSs) in vein graft re-endothelialization and remodelling in this study. METHODS AND RESULTS: Mouse isogenic vena cava was grafted into the carotid artery. Progressive remodelling of the grafted veins was evidenced by re-endothelialization at 2 weeks and subsequent appearance of vasomotor function at 4 weeks. Pharmacological inhibition of inducible NOS (iNOS) with the specific inhibitor 1400W, administered between 2 and 4 weeks after grafting, when re-endothelialization was complete, resulted in neoadventitial inflammation, neoadventitial thickening and impaired functional remodelling. CONCLUSION: Completion of re-endothelialization is pivotal in vein graft remodelling in the mouse and is associated with a series of changes in inflammation, proliferation and initiation of vascular functional remodelling. After re-endothelialization, iNOS upregulation may be an important mechanism to prevent secondary neoadventitial inflammation and preserve ongoing functional remodelling. iNOS activity could therefore be beneficial for long-term patency of the vein graft.

Angiotensin II differentially modulates cyclooxygenase-2, microsomal prostaglandin E2 synthase-1 and prostaglandin I2 synthase expression in adventitial fibroblasts exposed to inflammatory stimuli.

AIMS: To assess whether angiotensin II (Ang II) modulates key enzymes of the cyclooxygenase (COX)-2/prostanoid pathway, including prostaglandin E synthase-1 (mPGES-1) and prostacyclin synthase (PGIS) in rat aortic adventitial fibroblasts in the presence or absence of an inflammatory stimulus [interleukin (IL)-1ß]. METHODS AND RESULTS: Fibroblasts stimulated with IL-1ß (10 ng/ml, 24 h) and/or Ang II (0.1 µmol/l, 24 h) were used. IL-1ß up-regulated COX-2 and mPGES-1 (protein and mRNA) and increased PGI2 and PGE2 release, without altering PGIS protein expression. Ang II did modify neither COX-2 and mPGES-1 expression nor prostanoid levels, but it induced PGIS expression. Interestingly, Ang II further enhanced IL-1ß-induced COX-2 expression and PGI2 release and concomitantly reduced IL-1ß-induced mPGES-1 expression. The AT1 receptor antagonist losartan prevented the effects of Ang II on IL-1ß-induced COX-2 or mPGES-1 expression. IL-1ß activated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 pathways, and coincubation with Ang II resulted in a higher and more sustained phosphorylation of both MAPK. Inhibition of either p38 MAPK (SB203580) or ERK1/2 (PD98059) reduced COX-2 and mPGES-1 expression in cells treated with IL-1ß or the combination of IL-1ß and Ang II. Ang II did not modify COX-2 transcriptional activity but increased COX-2 mRNA stability in IL-1ß-treated cells; by contrast, it increased PGIS mRNA levels through a transcriptional mechanism. CONCLUSION: Ang II differentially modulates key enzymes involved in prostanoid biosynthesis thereby altering the balance between PGI2/PGE2 in vascular cells exposed to inflammatory stimuli.

Accelerated aging phenotype in mice with conditional deficiency for mitochondrial superoxide dismutase in the connective tissue.

The free radical theory of aging postulates that the production of mitochondrial reactive oxygen species is the major determinant of aging and lifespan. Its role in aging of the connective tissue has not yet been established, even though the incidence of aging-related disorders in connective tissue-rich organs is high, causing major disability in the elderly. We have now addressed this question experimentally by creating mice with conditional deficiency of the mitochondrial manganese superoxide dismutase in fibroblasts and other mesenchyme-derived cells of connective tissues in all organs. Here, we have shown for the first time that the connective tissue-specific lack of superoxide anion detoxification in the mitochondria results in reduced lifespan and premature onset of aging-related phenotypes such as weight loss, skin atrophy, kyphosis (curvature of the spine), osteoporosis and muscle degeneration in mutant mice. Increase in p16(INK4a) , a robust in vivo marker for fibroblast aging, may contribute to the observed phenotype. This novel model is particularly suited to decipher the underlying mechanisms and to develop hopefully novel connective tissue-specific anti-aging strategies.