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PubMed2005

Biochemical characterisation of the proteins encoded by the DiGeorge critical region 6 (DGCR6) genes.

The DiGeorge critical region 6 (DGCR6) gene exists in two highly homologous copies (DGCR6 and DGCR6L) on chromosome 22q11 and is deleted in patients with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). The DGCR6 mRNA levels are increased in metastatic mammary tumour cells and regulate the expression of neighbouring genes at the 22q11 region. Newly developed monoclonal antibodies detected predominantly nuclear phosphoproteins of approximately 25 kDa, with low expression levels in the cytoplasm. Both proteins have half-lives of about 2.5 h. Exogenously expressed DGCR6 and DGCR6L migrated with slightly different mobility in SDS-gels in accordance with two immunoreactive bands observed for the endogenous proteins. DGCR6 is found at low levels in primary human fibroblasts or peripheral blood mononuclear cells, while tumour cells, B-cells transformed by EBV as well as activated normal human T cells, contain elevated levels of the proteins. The proteins are differentially expressed in mammalian tissues, with high protein levels in heart, liver and skeletal muscle. These observations are important as some patients with DGCR6 syndrome exhibit a T-cell deficiency and/or cardiac malformations. As the DGCR6 protein(s) influence gene expression in trans, we analysed the influence of DGCR6/DGCR6L on the Epstein-Barr virus-encoded oncoproteins EBNA2 and EBNA3c in the activation of the viral LMP1 promoter, as well as LMP1-mediated activation of NFkB, but found no effect in either setting.

Animals↗
PubMed2001

The renin angiotensin system as a risk factor for coronary artery disease.

The renin angiotensin system was demonstrated to play a significant role in the genesis of hypertension and regulation of vascular tone over 100 years ago. The early investigations were subsequently expanded to implicate the renin angiotensin system in a variety of physiologic processes that may play a significant role in the initiation and progression of atherosclerosis. The renin angiotensin system modulates vascular structure and left ventricular hypertrophy via a number of trophic effects. Elevated levels of angiotensin II are associated with the generation of oxidative stress, and may thus play a significant role in the earliest phases of atherosclerosis. The role inflammation plays in atherosclerosis is amplified by the renin angiotensin system via the effects on adhesion molecules, growth factors, and chemoattractant molecules, which modulate the migration of inflammatory cells into the subendothelial space. The effects of angiotensin II, which may be at least partially genetically mediated, have been implicated in epidemiologic and clinical studies as a risk factor for the development of atherosclerosis. This review centers on the potential role that the renin angiotensin system plays as a risk factor for the development of atherosclerosis, and the role of converting enzyme inhibition or angiotensin receptor blockade as a mechanism to decrease the initiation, progression, and clinical consequences of the atherosclerotic process.

Coronary Disease↗
PubMed1988

Characterization of endosteal osteoblastic cells isolated from mouse caudal vertebrae.

We have developed a reliable procedure for isolating endosteal osteoblasts from mouse trabecular bone. Endosteal osteoblasts were obtained by migration and proliferation of the cells from the metaphyseal bone surface of caudal vertebrae onto nylon meshes. The isolated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cell population consisted of 95% alkaline-phosphatase-positive cells. The cell level of alkaline phosphatase was elevated (1.19 +/- 0.26 (SD) mumol PNP/mn/mg protein) and the enzyme activity was heat-inhibitable, indicating its skeletal origin. Light and electron microscopic observation revealed that cells have morphologic and ultrastructural appearance of typical osteoblasts with high protein synthesis activity. Osteoblasts grown in multilayers in the presence of 50 micrograms/ml ascorbic acid produced within 4 days an abundant fibrous intercellular collagenous matrix forming nodules in which osteocyte-like cells were embedded. Immunolabeling revealed synthesis of type I collagen but no detectable type III collagen. In presence of 7 mM beta-glycerophosphate the matrix became mineralized after 14-21 days of culture. Mineralization could not be induced by mouse skin fibroblasts cultured under similar conditions. The mineral deposits were closely associated with the collagen matrix, consisted of EDTA-removable, Von Kossa and alizarin red S stainable material and were composed of hydroxyapatite crystals identified by X-ray electron probe microanalysis. The isolated endosteal osteoblasts also displayed an intense (+457%) increase in intracellular cAMP production in response to human (1-34) PTH (2 x 10(-8) M) stimulation. The confluent cells responded to 20 nM 1,25(OH)2D3 by a significant 45% reduction in heat labile alkaline phosphatase activity. This procedure allowed us to isolate from trabecular bone a cell population that differentiates into osteoblasts in vitro, respond to calcitropic hormones and that retains its capacity to form a calcified bone tissue in culture. This method provided us a culture system for investigating the differentiation and metabolism of endosteal osteoblastic bone forming cells.

Animals↗
PubMed2005

Laminin gamma2-chain fragment circulating level increases in patients with metastatic pancreatic ductal cell adenocarcinomas.

Laminin-5 (LN5) is expressed solely by epithelial cells and considered to enhance cell migration after being cleaved by proteases, leading to the shedding of the N-terminal fragment of the LN5 gamma2-chain (G2F). We estimated circulating G2F level in 181 patients with various digestive diseases and 15 healthy subjects. The G2F level in pancreatic ductal cell adenocarcinoma patients with liver metastases was markedly elevated, but that in patients without liver metastases was not significantly elevated. The G2F levels in patients with benign pancreatic tumours (pancreatic cysts and intraductal papillary mucinous tumours) were similar to that in healthy volunteers. On the other hand, the level of the gamma1-chain is a common constituent of several laminin heterotrimers. The N-terminal fragment of gamma1-chain (G1F) in the circulation of these patients was also determined, and a slight increase in G1F levels was observed only in hepatocellular carcinoma patients. Interestingly, a significant increase in circulating G2F/G1F ratio was observed in patients with bile duct and gallbladder carcinoma, as well as in those with metastatic pancreatic ductal cell adenocarcinoma. The increase in the circulating G2F level, particularly compared with circulating G1F level, should correlated with LN5 overexpression in invasive carcinomas, independently of basement membrane metabolism in the entire body.

Adenocarcinoma↗
PubMed2005

Dynamics of antibodies against hypodermin C in reindeer infested with the reindeer warble fly, Hypoderma tarandi.

Serum samples from 25 reindeer (Rangifer tarandus tarandus) were assayed for antibody against hypodermin C (HyC) using an ELISA. Nineteen animals were calves (born in 1998, 1999 or 2001) and six were adults (3-10 years old at first blood collection). The samples were collected over periods of 4 months (calves born in 2001) or 27 months (adults and calves born in 1998 and 1999), the latter encompassing three Hypoderma tarandi infestation seasons. The calves received antibodies against HyC from their mothers, either by placental transfer or through the ingestion of colostrum. The low level at 3 h postpartum compared to the high level 3 days after birth in one calf suggests that the antibodies are transferred through colostrum. The levels of antibody of maternal origin decreased rapidly and reached low levels by mid-July, which coincides with the onset of the major Hypoderma ovipositioning season in this region. The calves thus did not appear to be protected by antibody against HyC when they were exposed to H. tarandi infestation for the first time. Antibody levels increased following infestation and reached a maximum during November or December, which coincides with when the H. tarandi larva stops migrating after it has reached the site under the skin of the back of the host and develops further. Levels declined thereafter and reached a nadir during the following summer. After the subsequent re-infestation, the increase in levels occurred at least 1 month earlier than with the first infestation. Levels remained elevated throughout the year after repeated infestations. This implies that the antibodies persist after the annual exit of mature larvae from the animal, and after larvae have been killed by application of ivermectin. Levels in adults, however, declined significantly with age, and levels were significantly lower in animals that were 4-11 years old than in 1-year-old animals during the same 1-year period. This supports the contention that the functional capacity of the immune system declines gradually with age. The study demonstrated that HyC is potentially useful for serological diagnosis of hypodermosis in reindeer, but the persistence of antibodies complicates interpretation of antibody-based surveillance programme data in all cases other than first-time exposure.

Animals↗
PubMed2004

Regulation of expression of the Na+/H+ exchanger by thyroid hormone.

The Na+/H+ exchanger is a pH regulatory protein with a ubiquitous distribution in eukaryotic cells. Several isoforms of the Na+/H+ exchanger are known. The first isoform to be characterized and cloned, NHE1, is present on the plasma membrane of cells and functions to remove one intracellular proton in exchange for one extracellular sodium ion. It is involved in pH regulation, cell growth, differentiation, and cell migration. NHE1 is also involved in the cycle of damage that occurs in the heart with ischemic heart disease. Recent studies have shown that the Na+/H+ exchanger is regulated in response to thyroid hormone. Reduction in circulating thyroid hormone levels reduces the amount of both protein and mRNA of NHE1. Conversely, an elevation of thyroid hormone levels has the opposite effects. Transcriptional regulation of NHE1 expression has been demonstrated. The NHE1 promoter contains a TR alpha(1) binding site located between -841 to -800 bp. This element responds positively to TR alpha(1). This regulation of the NHE1 promoter by thyroid hormone is proposed to be responsible for postnatal changes in expression of the Na+/H+ exchanger.

Animals↗
PubMed2002

Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections.

Dendritic cells (DC) accumulate in the CNS during inflammation and may contribute to local immune responses. Two DC subsets present in human cerebrospinal fluid (CSF) are probably recruited from myeloid (CD11c(+)CD123(dim)) and plasmacytoid (CD11c(-)CD123(high)) blood DC. In bacterial meningitis and especially in Lyme meningoencephalitis, numbers of myeloid and plasmacytoid DC in CSF were increased, compared to non-inflammatory neurological diseases, and correlated with chemotactic activity of CSF for immature monocyte-derived DC (moDC). Multiple DC chemoattractants, including macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-1, MCP-3, RANTES and stromal cell-derived factor (SDF)-1alpha were elevated in CSF in these two neuroinfections. Chemotaxis of immature moDC induced by these CSFs could be partially inhibited by mAbs against CXCR4, the receptor for SDF-1alpha, and CD88, the receptor for C5a. SDF-1alpha present in CSF also chemoattracted mature moDC, which in vivo could correspond to a diminished migration of antigen-bearing DC from the CSF to secondary lymphoid organs. Regulation of DC trafficking to and from the CSF may represent a mechanism of controlling the CNS inflammation.

Adolescent↗
PubMed2000

Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large docking protein Gab1 and the adapter protein CRKL.

Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis.

Matrix metalloproteinases (MMPs) are implicated in multiple sclerosis where one of their roles may be to facilitate the transmigration of circulating leukocytes into the CNS. Studies have focused on only a few MMPs, and much remains unknown of which of the 23 MMP family members is/are critical to the multiple sclerosis disease process. Using quantitative real time polymerase chain reactions, we have systematically analysed the expression of all 23 MMP members in subsets of leukocytes isolated from the blood of normal individuals. We found a distinctive pattern of MMP expression in different cellular populations: MMP-11, MMP-26 and MMP-27 were enriched in B cells, while MMP-15, MMP-16, MMP-24 and MMP-28 were prominent in T lymphocytes. Of interest is the enrichment of a majority of MMP members in monocytes: MMP-1, MMP-3, MMP-9, MMP-10, MMP-14, MMP-19 and MMP-25. MMP-2 and MMP-17 were also significantly represented in monocytes, although B cells had significant amounts of these MMPs. In correspondence with their strong expression of many MMP members, monocytes migrated more rapidly across a model of the blood-brain barrier in culture than T or B lymphocytes. Finally, we found higher levels of two of the monocyte-expressed MMPs in multiple sclerosis patients compared with normal individuals: MMP-2 and MMP-14. Tissue inhibitor of metalloproteinases (TIMP)-2 was also elevated in monocytes from multiple sclerosis patients, providing a mechanism for the reported activation of MMP-2 by MMP-14 and TIMP-2. These results emphasize that monocytes are prominent contributors of the neuroinflammation in multiple sclerosis through a mechanism that involves their high MMP expression and that they identify specific MMP members as targets for novel therapeutics in the disease.

Adult↗
PubMed2000

Quantitative analysis of fushi tarazu factor 1 homolog messenger ribonucleic acids in the pituitary of salmon at different prespawning stages.

Steroidogenic factor 1 (SF-1) or Ad4BP is a member of the fushi tarazu factor 1 (FTZ-F1) family and an orphan nuclear receptor that plays an important role in the hypothalamus-pituitary-gonadal axis and the adrenal cortex. Although its critical role in the differentiation of adrenals, gonads, and pituitary gonadotropes has been well demonstrated, regulatory function of SF-1 during sexual maturation is yet to be examined. To investigate the potential role of SF-1 in sexual maturation, expression of two salmon FTZ-F1 homolog genes, sFF1-I and sFF1-II, was examined in the pituitaries of chum and sockeye salmons, using specific and sensitive RNase protection assays. Only sFF1-I mRNA was found in the pituitary and other organs, such as the ovary, spleen, liver, brain, and skeletal muscle. In chum salmon during upstream migration from the bay to the hatchery, the level of sFF1-I mRNA in the male fish was increased on the midway in the river, where the levels of gonadotropin alpha- and II beta-subunit mRNAs were increased. In maturing sockeye salmon, the expression of the sFF1-I gene was elevated in the mature male fish, but the administration of GnRH analog did not further enhance the expression. These results indicate that sFF1-I gene expression in the pituitary is upregulated in maturing salmon, and this upregulation may not depend on GnRH.

Animals↗
PubMed1982

Role of platelets and fibrin in the healing sequence: an in vivo study of angiogenesis and collagen synthesis.

UNLABELLED: The signals that initiate repair are poorly characterized. These studies investigate the capacity of platelets and fibrin to initiate angiogenesis, fibroplasia, collagen synthesis and monocyte migration in the rabbit cornea assay. METHODS: Autologous platelets and platelet-free fibrin were isolated from rabbit blood. Released and control platelet preparations and autologous and commercial fibrin were implanted in rabbit corneas. RESULTS: Thrombin-released platelets produced angiogenesis and opacification. Histology showed fibroplasia, corneal thickening, and neovascularization. Collagen synthesis was elevated to twice control levels in thrombin-activated platelet preparations. Various control platelet preparations produced no angiogenesis, no opacification, and no histologic change. All fibrin injections elicited a cellular exudate from the limbal vessels, followed by angiogenesis and corneal opacification. Histology showed a mononuclear infiltrate with neovascularization and fibroplasia. Control injections of rabbit skin collagen and fibroblasts produced no response.

Animals↗
PubMed2001

Estrogen receptor-mediated, nitric oxide-dependent modulation of the immunologic barrier function of the endothelium: regulation of fas ligand expression by estradiol.

BACKGROUND: Premenopausal women have a lower incidence of coronary artery disease than postmenopausal women or same-age men. Although the mechanisms of this apparent relative protection against atherosclerosis remain ill defined, estradiol, which is present in higher concentrations before menopause, is considered to play a central role. Recently, Fas ligand (FasL) expression by the vascular endothelium has been shown to inhibit the migration of inflammatory cells into the vessel wall, an event that is considered crucial for the development of atherosclerosis. METHODS AND RESULTS: The regulation of endothelial FasL expression by estradiol was investigated in vivo and in vitro. In an ovariectomized, cholesterol-clamped rabbit model, FasL expression was shown to be downregulated by elevations in serum cholesterol, which also resulted in invasion of the arterial wall by macrophages. Estradiol replacement resulted in restoration of FasL expression, with resultant inhibition of leukocyte traffic across the endothelium. Inhibition of NO production by addition of L-NAME to the drinking water of the estradiol-treated rabbits abrogated these effects. In vitro, estradiol is shown to regulate FasL expression at the transcriptional level via an estrogen receptor-mediated, NO-dependent mechanism. CONCLUSIONS: Estradiol transcriptionally regulates endothelial FasL expression by a mechanism involving at least one of the estrogen receptors. In an animal model of atherosclerosis, estradiol restores FasL expression, which is suppressed by atherogenic levels of serum cholesterol. The maintenance of endothelial FasL expression by estradiol may represent a mechanism of estrogen's apparent antiatherogenic effect.

Animals↗
PubMed2001

Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis.

Excess fibroblasts and inflammatory cells may play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The small GTPase, Rho, and its target protein, Rho-associated coiled-coil-forming protein kinase (ROCK), have been recognized to be major regulators of cell locomotion mediated by reorganization of the actin cytoskelton. Activated ROCK inhibits myosin phosphatase, and this in turn induces phosphorylation of the myosin light chain (MLC). To determine the mechanisms underlying the deterioration process of IPF, we investigated the effect of Y-27632, a selective ROCK inhibitor, in a murine model of bleomycin (BLM)-induced lung fibrosis. The Aschcroft score and hydroxyproline content of the BLM-treated mouse lung decreased in response to Y-27632 treatment. The number of broncoalveolar cells was decreased by Y-27632, and migration of macrophages, neutrophils, and fibroblasts in vitro was inhibited by Y-27632 regardless of various stimuli. Although expression of ROCK-II mRNA in the lung homogenates of the BLM-treated mice was increased approximately 9-fold, expression of ROCK-II protein showed only a slight tendency to increase. BLM elevated MLC phosphorylation levels, and Y-27632 inhibited BLM response. These findings indicate that the Rho/ROCK-mediated pathway plays an important role in IPF, and that blocking of this pathway leads to inhibition of IPF development.

Animals↗
PubMed2001

Neonatal T cells in an adult lung environment are competent to resolve Pneumocystis carinii pneumonia.

Initiation of the pulmonary inflammatory response to Pneumocystis carinii is delayed by 3 wk in mice infected as neonates compared with adults. There was no difference in the proliferative response of draining lymph node T cells from mice infected as neonates compared with adults when stimulated in vitro with either Con A or anti-CD3 mAB: However, TNF-alpha and IFN-gamma mRNA expression in the lungs of P. carinii-infected neonates was significantly lower than in adults indicating a lack of appropriate activation signaling in the local environment. This may have been due to active suppression because TGF-beta mRNA expression was significantly elevated in neonatal lungs compared with adults. To determine whether T cells from 10-day-old mice would effect resolution of P. carinii if harbored in an adult lung environment, cells were adoptively transferred to SCID mice with established P. carinii infections. There was no difference in the kinetics of T cell migration into the lungs or of clearance of P. carinii organisms when SCID mice were reconstituted with splenocytes from young mice as compared with adult mice. Furthermore, splenocytes from young mice stimulated both TNF-alpha and IFN-gamma mRNA expression to levels that were similar to that in the lungs of SCID mice reconstituted with adult cells. These data indicate that neonatal lymphocytes are competent to resolve P. carinii infection when harbored in an adult lung environment, suggesting that the neonatal lung environment, and not the T cells, is ineffective at responding to P. carinii infection.

Aging↗
PubMed1999

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.

Apoptosis↗
PubMedDate not supplied

Alteration of DNA base excision repair enzymes hMYH and hOGG1 in hydrogen peroxide resistant transformed human breast cells.

BACKGROUND: Oxidative stress is a major causative agent of carcinogenesis, aging, and a number of diseases. 8-oxoG is the most stable and deleterious lesion of oxidative DNA damage. The 8-oxoG lesions can be eliminated by human repair systems consisting of three enzymes hMTH1, hOGG1, and hMYH homologous to E. coli MutT, MutM, and MutY proteins, respectively. MATERIAL AND METHODS: Human cells (P1, P2, and P3) resistant to H(2)O(2) were derived from the non-tumorigenic human breast cell line MCF10A by sequential treatment of the cells with H(2)O(2). The protein expression levels of DNA repair enzymes were analyzed by Western blotting. The DNA binding and glycosylase activities of hMYH and hOGG1 were measured in the extracts of the H(2)O(2) resistant cells. RESULTS: The H(2)O(2) resistant cells displayed tremendously greater anchorage-independent growth capability and higher expression of the anti-apoptotic protein BCL-2 than the parental cells. H(2)O(2) detoxification ability was elevated in P1 and P2 cells, but not in P3 cells, suggesting P3 cells might employ a different defense mechanism from P1 and P2 cells. In P3 cells, both hOGG1 and hMYH glycosylase activities were reduced but their protein levels increased. Two A/8-oxoG binding complexes were detected with cell extracts: the fast-migrating complex (bottom form) was dominated in MCF10A cells, and was greatly reduced in P3 cells. Interesting, the P3 cells showing the least amount of bottom form had the weakest hMYH glycosylase activity. CONCLUSIONS: Our results demonstrated, for the first time, that alteration of base excision repair pathways is correlated to cell resistance to oxidative stress.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Matrix metalloproteinase-2 is elevated in the plasma of women with preeclampsia.

OBJECTIVE: We have recently demonstrated that matrix metalloproteinase-2 (MMP-2) alters vascular function through cleavage of vasoactive peptides, resulting in increased vasoconstriction and reduced vasodilation. We, therefore, hypothesized that MMP levels are increased in women with preeclampsia. In addition, because vascular endothelial growth factor (VEGF) has been implicated in the pathophysiology of preeclampsia and is involved in angiogenesis that requires the release of proteases to allow for migration of endothelial cells, we hypothesized that VEGF increases release of MMPs from endothelial cells. METHODS: We used zymographic analysis to evaluate MMP-2/MMP-9 levels in plasma of women with preeclampsia (n=12) compared to women with uncomplicated pregnancies (n=12). In addition, we evaluated the changes in the levels of MMP-2 and MMP-9 as well as tissue inhibitors of MMPs (TIMP-1 and TIMP-2) released by cultured human umbilical vein endothelial cells in response to VEGF (0.1-10 ng/mL). RESULTS: Our data showed that plasma MMP-2 levels were significantly higher in women with preeclampsia compared to women with uncomplicated pregnancies (arbitrary intensity units: 690 +/-111 and 252 +/-56, respectively, p<0.05). MMP-9 levels were below the level of detection. In addition, VEGF stimulated endothelial MMP-2 and MMP-9 release in a concentration- and time-dependent (6-24 h) manner. Moreover, VEGF stimulation of MMP release occurs without significantly affecting the release of TIMP-1 and TIMP-2. CONCLUSIONS: These data suggest that VEGF promotes secretion of MMPs from endothelial cells that, in turn, could alter vascular function in women with preeclampsia.

Adult↗
PubMed1992

Alcohol dose and low density lipoprotein heterogeneity in squirrel monkeys (Saimiri sciureus).

The present study was designed to determine whether normolipidemic male squirrel monkeys (Saimiri sciureus) exhibit low density lipoprotein (LDL) heterogeneity similar to that observed in humans and if present, whether LDL subfractions are altered by consumption of low vs. high dose ethanol (EtOH). Primates were divided into three groups designated control, low, and high EtOH and fed isocaloric liquid diets containing 0%, 12% and 24% of calories as EtOH, respectively, for 6 months. The 12% EtOH caloric level resulted in a modest, non-significant increase in high density lipoprotein (HDL) cholesterol and no change in LDL cholesterol or plasma apolipoprotein B (apo B), while the 24% dose produced significant elevations in plasma, LDL and HDL cholesterol and apo B. Using a single-spin density gradient ultracentrifugation procedure developed for humans, three distinct LDL subclasses designated LDL1a (d = 1.031 g/ml), LDL1b (d = 1.038 g/ml) and LDL 2 (d = 1.046 g/ml) were isolated from all three treatment groups. Monkey LDL subfractions were nearly identical to very light, light and heavy LDL subspecies isolated from human plasma in terms of their: (1) isopycnic densities following ultracentrifugation; (2) co-migration as single bands with beta-electrophoretic mobility in cellulose acetate and agarose electrophoretic gels; (3) size-dependent migration pattern in polyacrylamide gradient electrophoretic gels; (4) co-migration as a single band corresponding to apo B-100, following SDS polyacrylamide gel electrophoresis; and (5) decrease in total cholesterol/protein ratios with increasing LDL subclass density. Although there were no treatment differences in LDL particle size, within each treatment group, mean particle size for each LDL subfraction was significantly different from every other subfraction. Low (12%) dose alcohol had no effect on LDL subfraction mass relative to controls while high alcohol consumption resulted in marked increases in all lipid (except triglyceride) and protein of the larger, buoyant LDL subspecies (LDL1a and LDL1b). Moreover, the best correlation between plasma apo B and LDL subfraction total mass was demonstrated with LDL1b (r = 0.735). Since neither the lipid nor the protein concentration of the small, dense, purportedly more atherogenic, LDL2 changed with the 24% EtOH dose, we propose that the LDL subfraction alterations associated with high alcohol intake in squirrel monkeys (increased LDL1a, increased LDL1b, LDL2 no effect) may represent a compensatory response to modulate the overall atherogenic lipoprotein profile associated with elevations in total LDL cholesterol and plasma apolipoprotein B.

Animals↗