Secretions of MMP-9 by soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) mediated by protein kinase C (PKC)delta and phospholipase D (PLD) in murine macrophage

The secretion of matrix metalloproteinase (MMP-9) is stimulated by the glucocorticoid-induced tumor necrosis factor receptor (GITR), a new tumor necrosis factor receptor (TNFR) family, in murine macrophages via an activation of protein kinase C (PKC)delta and phospholipase D (PLD). Secretions of MMP-9 are stimulated by the phosphatidic acid (PA), a product of PLD activity and an inhibition of PA production by a 1-propanol inhibited secretion of MMP-9 by soluble GITR (sGITR). MMP-9 is not secreted by diacylglycerol (DAG) and an inhibitor of PA phosphatase has no effect on the secretion induced by sGITR, indicating that PA is responsible for MMP-9 secretion in murine macrophages. Our data indicates that sGITR-induced activation of PKCdelta and PLD increases MMP-9 secretions in macrophages.     

1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.     

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.     

Role of PTHrp and PTHrp-engaged pathways in MCF-7 cells migration/invasion.

In this paper the effect of N-terminal parathyroid hormone-related protein (PTHrp) and PTHrp-engaged pathways on MCF-7 breast cancer cell migration/invasivity and matrix metalloproteinases (MMPs) production were investigated. We found that: a) migration is not affected by PTHrp and Forskolin (FK)-activated PKA, while Phorbol Myristate Acetate (PMA)-activated PKC strongly stimulates MCF-7 cells motility. b) MMPs production was unaffected by PTHrp, but FK reduced membrane-type (MT)-1 MMP expression. Conversely, PMA induced a marked increase of MT1-MMP and MMP-9. c) Chemical activation of PKC is not sufficient, by itself, to confer invasive ability to MCF-7 cells, unless they were provided with additional factors, supplied by fibroblasts. d) Matrix invasion likely occurs through an activation cascade, involving at least three components: pro-MMP-9 and MT-1 MMP (supplied by PMA-stimulated MCF-7 cells) and pro MMP-2 (supplied by fibroblasts). e) The selective chemical inhibition of the adenylylciclase (AC)/PKA and phospholipase C (PLC)/PKC pathways confirmed that MCF-7 cells invasivity is not affected by exogenous PTHrp, which can only modulate their growth. However, the PTHrp responsibility in breast cancer invasion cannot be completely excluded. Indeed, fibroblasts are known to respond to PTHrp (which is a normal product of MCF-7 as well as other breast cancer cells) with enhanced release of MMP-2. On the basis of the documented requirement of fibroblast-derived MMP-2 for MCF-7 cell invasivity, a novel humoral fibroblast-breast cancer cell interaction, mediated by PTHrp, can be recognised.     

Liberation of desmosine and isodesmosine as amino acids from insoluble elastin by elastolytic proteases

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Membrane mediated signaling mechanisms are used differentially by metabolites of vitamin D(3) in musculoskeletal cells

1 alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) mediate their effects on chondrocytes and osteoblasts in part through increased activity of protein kinase C (PKC). For both cell types, 1 alpha,25(OH)(2)D(3) exerts its effects primarily on more mature cells within the lineage, whereas 24R,25(OH)(2)D(3) exerts its effects primarily on relatively immature cells. Studies using the rat costochondral cartilage growth plate as a model indicate that the two metabolites increase PKC activity by different mechanisms. In growth zone cells (prehypertrophic/upper hypertrophic cell zones), 1 alpha,25(OH)(2)D(3) causes a rapid increase in PKC that does not involve new gene expression. 1 alpha,25(OH)(2)D(3) binds its membrane receptor (1,25-mVDR), resulting in activation of phospholipase A(2) and the rapid release of arachidonic acid, as well as activation of phosphatidylinositol-specific phospholipase C, resulting in formation of diacylglycerol and inositol-1,4,5-tris phosphate (IP(3)). IP(3) leads to release of intracellular Ca(2+) from the rough endoplasmic reticulum, and together with diacylglycerol, the increased Ca(2+) activates PKC. PKC is then translocated to the plasma membrane, where it initiates a phosphorylation cascade, ultimately phosphorylating the extracellular signal-regulated kinase-1 and -2 (ERK1/2) family of MAP kinases (MAPK). PKC increases are maximal at 9 min, and MAPK increases are maximal at 90 min in these cells. By contrast, 24R,25(OH)(2)D(3) increases PKC through activation of phospholipase D in resting zone cells. Peak production of diacylglycerol via phospholipase D2 is at 90 min, as are peak increases in PKC. Some of the effect is direct on existing plasma membrane PKC, but most is due to new PKC expression; translocation is not involved. Arachidonic acid and its metabolites also play differential roles in the mechanisms, stimulating PKC in growth zone cells and inhibiting PKC in resting zone cells. 24R,25(OH)(2)D(3) decreases phospholipase A(2) activity and prostaglandin production, thereby overcoming this potential inhibitory component, which may account for the delay in the PKC response. Ultimately, ERK1/2 is phosphorylated. PKC-dependent MAPK activity transduces some, but not all, of the physiological responses of each cell type to its respective vitamin D metabolite, suggesting that the membrane receptor(s) and nuclear receptor(s) may function interdependently to regulate proliferation and differentiation of musculoskeletal cells, but different pathways are involved at different stages of phenotypic maturation.     

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices.

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Elasto-regenerative properties of polyphenols

Abdominal aortic aneurysms (AAA) are progressive dilatations of infra-renal aorta causing structural weakening rendering the aorta prone to rupture. AAA can be potentially stabilized by inhibiting inflammatory enzymes such as matrix metalloproteinases (MMP); however, active regression of AAA is not possible without new elastic fiber regeneration. Here we report the elastogenic benefit of direct delivery of polyphenols such as pentagalloyl glucose (PGG), epigallocatechin gallate (EGCG), and catechin, to smooth muscle cells obtained either from healthy or from aneurysmal rat aorta. Addition of 10 μg/ml PGG and ECGC induce elastin synthesis, organization, and crosslinking while catechin does not. Our results indicate that polyphenols bind to monomeric tropoelastin and enhance coacervation, aid in crosslinking of elastin by increasing lysyl oxidase (LOX) synthesis, and by blocking MMP-2 activity. Thus, polyphenol treatments leads to increased mature elastin fibers synthesis without increasing the production of intracellular tropoelastin.     

Calcitonin gene-related peptide released from endothelial progenitor cells inhibits the proliferation of rat vascular smooth muscle cells induced by angiotensin II

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.     

Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats

Polycystic ovary syndrome (PCOS) is characterized by cystogenesis; however, the cause of this cystogenesis is unknown. At ovulation, preovulatory collagenolytic activities in the ovarian follicles increase and various proteinases are needed to degrade the tissues surrounding the follicles. To clarify the roles of enzymes in collagen degradation of the follicular wall of polycystic ovary (PCO) in relation to the cystogenesis, we examined expression of lysyl oxidase (LOX), which initiates cross-link formation of the collagen and elastin in the extracellular matrix, and expression of matrix metalloproteinases (MMPs) in ovaries of model rats with PCO induced by dehydroepiandrosterone (DHEA) compared with MMP expression in control rats. DHEA treatment increased LOX mRNA expression to more than three times the control value (P: < 0.01). MMP-2 mRNA expression in control rats was threefold greater than that in the DHEA-induced group (P: < 0.05). Expression of both latent and active forms of MMP-2 in controls was more than twice that in the DHEA-induced group (P: < 0.05) as shown by Western blotting, and expression of the active form of MMP-2 was also twice as high in the controls as in the DHEA-treated group (P: < 0.05) as shown by zymography. Our results suggest that depression of MMP-2 activity and increased LOX expression may be one of the causes of the cystogenesis of PCO.     

Homocysteine decreases blood flow to the brain due to vascular resistance in carotid artery

An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine β Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS−/−) die early, but heterozygous for (CBS−/+) survive with high levels of Hcy. The γ-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA_A receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS−/+, CBS−/+ GABA_A−/− double knockout, and GABA_A−/− were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS−/+/GABA_A−/−double knockout > CBS−/+/ > GABA_A−/− compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA_A−/−, CBS−/+, CBS−/+/GABA_A double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.     

Inhibition of phospholipases by Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe, C terminus of middle-sized tumor antigen

The N and C terminals and tyrosine-phosphorylating site of the middle-sized tumor antigen of polyoma virus were chemically synthesized. The sequences of these peptides were Met-Asp-Arg-Val-Leu-Ser-Arg-Ala-Asp-Lys (N-MT), Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe (C-MT), and Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (MT-Tyr), respectively. Among these peptides, the C-MT peptide inhibited phospholipase A2 (EC 3.1.1.4), phospholipase C (EC 3.1.4.3), and phospholipase D (EC 3.1.4.4). In addition, phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) was also inhibited by this peptide. To study the mechanism of the inhibition, kinetic analysis was performed using phospholipase A2 from porcine pancreas. The degree of inhibition of phospholipase was dose dependent, and maximal inhibition was observed at pH 8.8. This peptide inhibited phospholipase A2 in a competitive manner for low-affinity sites of Ca2+, and in a noncompetitive manner for phospholipid substrates. When a fatty acid in the 2 position of the glycerol moiety of phosphatidylcholine was replaced by palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), eicosatrienoic acid (C20:3), or arachidonic acid (C20:4), the degree of inhibition of phosphatidylcholine hydrolysis by the C-MT peptide decreased. Inhibition of phospholipase A2 by the C-MT peptide was reversed by low concentrations of sodium deoxycholate but not by Triton X-100 or Nonidet P40, nonionic detergents. These detergents and the modification of acyl groups altered the micellar state of phospholipids. These results, taken together, suggest that the binding of the C-MT peptide near the low-affinity Ca2+ binding sites modifies the interaction of phospholipid substrates with the active center of phospholipase A2.     

Rational design of tropoelastin peptide-based inhibitors of metalloproteinases

Abnormal production of matrix metalloproteinases (MMPs) has been observed in a variety of diseases, such as emphysema, atherosclerosis, and cancer metastasis. Destruction of connective tissue ensues and elastin is often a key target. Three of the main elastolytic MMPs are the gelatinases MMP-2 and MMP-9 and the metalloelastase MMP-12. To investigate the possibility of using peptides to inhibit the elastolytic activity of these enzymes, we mapped the sites within tropoelastin recognized by MMP-9 and MMP-12. Peptides that correspond to regions overlapping these sites were then tested for their ability to inhibit these MMPs. These included an unmodified peptide directed against MMP-9 (peptide PP), cysteine-containing peptides that mimicked either the MMP-9 (peptide NCP) or the MMP-12 (peptide lin24) cleavage sites in tropoelastin and their cyclized forms (CP and cyc24, respectively), and a peptide containing a zinc-chelating hydroxamate group directed against MMP-9 (HP). The presence of a free sulfhydryl or hydroxamate group capable of chelating the zinc ion in the active site of the MMPs was generally found to increase the inhibitory activity of the peptides. The specificity of the inhibitors varied, with some of the inhibitors showing activity against all of the MMPs examined. None of the inhibitors had any significant effect on the activity of the unrelated serine protease, plasmin. K(i) values for the inhibitors were in the micromolar range. Our results suggest ways of developing other MMP inhibitors based on substrate recognition sites that may provide greater levels of inhibition.