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Biomedical subjects

M A Moses

Publications and source records attributed to M A Moses.

At least 37 records · Page 2Linked to original sources
PubMed1999

Troponin I is present in human cartilage and inhibits angiogenesis.

Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo.

3T3 Cells↗
PubMed1999

Tissue engineering of autologous aorta using a new biodegradable polymer.

BACKGROUND: Ovine pulmonary valve leaflets and pulmonary arteries have been tissue-engineered (TE) from autologous cells and biodegradable polyglycolic acid (PGA)-polyglactin copolymers. Use of this cell-polymer construct in the systemic circulation resulted in aneurysm formation. This study evaluates a TE vascular graft in the systemic circulation which is based on a new copolymer of PGA and polyhydroxyalkanoate (PHA). METHODS: Ovine carotid arteries were harvested, expanded in vitro, and seeded onto 7-mm diameter PHA-PGA tubular scaffolds. The autologous cell-polymer vascular constructs were used to replace 3-4 cm abdominal aortic segments in lambs (group TE, n = 7). In a control group (n = 4), aortic segments were replaced with acellular polymer tubes. Vascular patency was evaluated with echography. All control animals were sacrificed when the grafts became occluded. Animals in TE group were sacrificed at 10 days (n = 1), 3 (n = 3), and 5 months (n = 3). Explanted TE conduits were evaluated for collagen content, deoxyribonucleic acid (DNA) content, structural and ultrastructural examination, mechanical strength, and matrix metalloproteinase (MMP) activity. RESULTS: The 4 control conduits became occluded at 1, 2, 55, and 101 days. All TE grafts remained patent, and no aneurysms developed by the time of sacrifice. There was one mild stenosis at the anastomotic site after 5 months postoperatively. The percent collagen and DNA contents approached the native aorta over time (% collagen = 25.7%+/-3.4 [3 months] vs 99.6%+/-11.7 [5 months], p < 0.05; and % DNA = 30.8%+/-6.0 [3 months] vs 150.5%+/-16.9 [5 months], p < 0.05). Histology demonstrated elastic fibers in the medial layer and endothelial specific von Willebrand factor on the luminal surface. The mechanical strain-stress curve of the TE aorta approached that of the native vessel. A 66 kDa MMP-2 was found in the TE and native aorta but not in control group. CONCLUSIONS: Autologous aortic grafts with biological characteristics resembling the native aorta can be created using TE approach. This may allow the development of "live" vascular grafts.

Animals↗
PubMed1999

Molecular angiogenesis.

New insights into the mechanisms by which blood vessels develop (angiogenesis) have been gained recently, primarily by the identification of factors that inhibit and promote this process. Angiogenesis-stimulating factors are being used to promote growth of new blood vessels in ischemic disease. In contrast, anti-angiogenesis factors are being used as inhibitors of neovascularization to control tumor growth and metastasis.

Animals↗
PubMed1999

Fetal partial urethral obstruction causes renal fibrosis and is associated with proteolytic imbalance.

PURPOSE: We determine whether fetal bladder outlet obstruction induces renal fibrosis, and is associated with an alteration in the regulation of connective tissue degradation and the presence of fibrogenic interstitial cells. MATERIALS AND METHODS: Partial bladder outlet obstruction was surgically induced in 33 fetal sheep at 95 days of gestation. These animals and 24 normal age matched controls were sacrificed at 109, 116 and 135 (term) days of gestation, and the kidneys were rapidly retrieved, drained and weighed. Representative whole kidney samples were snap frozen for assessment of deoxyribonucleic acid, protein and collagen content. Morphometric analysis and alpha-smooth muscle actin immunohistochemistry were performed on histological specimens from formalin fixed kidneys. Tissue extract from fresh kidney specimens were analyzed for metalloproteinase and tissue inhibitor of metalloproteinase activity. Urine samples obtained at the time of sacrifice were analyzed for electrolyte, creatinine and N-acetyl glucosaminidase excretion. RESULTS: All obstructed kidneys were hydronephrotic and larger than age matched controls. Obstructed kidneys at term showed interstitial fibrosis, as measured by increased extracellular matrix volume fraction (45% in male obstructed kidneys versus 2.5% in normal male kidneys, p = 0.0004), increased total collagen content (120 mg./kidney in male obstructed versus 20 mg. in normal male animals, p = 0.016) and collagen/deoxyribonucleic acid content per kidney (2.78 versus 0.53 mg./mg., p = 0.016). Metalloproteinase-1 activity was significantly lower in obstructed kidneys (210 versus 380 U./mg. protein in normal kidneys). Tissue inhibitor of metalloproteinase activity was undetectable in both groups. The presence of an increased population of myofibroblasts often associated with fibrotic processes was seen by alpha-smooth muscle actin staining which was localized to interstitial cells throughout the cortex in obstructed kidneys. CONCLUSIONS: Fetal partial bladder outlet obstruction induces renal interstitial fibrosis as early as 2 weeks after obstruction. A possible mechanism for this process is a shift in proteolytic activity to reduce matrix degradation in obstructed kidneys. These changes might be mediated by the increased number of fibrogenic interstitial cells. The observations suggest several potential approaches to developing an understanding of congenital obstructive uropathy.

Actins↗
PubMed1999

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro.

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

Basement Membrane↗
PubMed1998

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C.

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC.

Alkaline Phosphatase↗
PubMed1998

Increased incidence of matrix metalloproteinases in urine of cancer patients.

Matrix metalloproteinases (MMPs) have been implicated in mechanisms of metastasis in experimental cancer models and in human malignancies. In this study, we used substrate gel electrophoresis (zymography) to determine the frequency of detection of MMPs in urine of patients with a variety of cancers. Three molecular weight classes of urinary MMPs, Mr 72,000, Mr 92,000, and high molecular weight (Mr > or = 150,000) species, were detected reproducibly and correlated with disease status. The Mr 72,000 and Mr 92,000 species were identified as MMP-2 and MMP-9, respectively, by Western blot analysis. The presence of biologically active MMP-2 (P < 0.001) or MMP-9 (P = 0.002) was an independent predictor of organ-confined cancer, and the high molecular weight species (P < 0.001) was an independent predictor of metastatic cancer. This is the first study to demonstrate that analysis of urinary MMPs may be useful in determining disease status in a variety of human cancers, both within and outside of the urinary tract.

Adult↗
PubMed1998

Molecular cloning and expression analysis of the cDNA encoding rat tissue inhibitor of metalloproteinase-4.

We have cloned and sequenced the cDNA encoding the rat tissue inhibitor of metalloproteinase 4 (TIMP-4). As a first step towards exploring the role of TIMP-4 in both the physiological and the pathological remodeling of extracellular matrix, we have extensively analyzed the mRNA expression of TIMP-4 in various rat tissues. The results reveal that the expression pattern of rat TIMP-4 is distinct from that of its human and mouse counterparts (Greene et al., 1996; Leco et al., 1997). In this report, we show that rat TIMP-4 is expressed much more extensively than previously reported, which suggests that it may play a significant role in modulating proteolysis of extracellular matrix in a wide range of tissue settings.

Amino Acid Sequence↗
PubMed1997

Matrix metalloproteinase-3 releases active heparin-binding EGF-like growth factor by cleavage at a specific juxtamembrane site.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored precursor that is cleaved to release the soluble mature growth factor. The two forms are active as juxtacrine and paracrine/autocrine growth factors, respectively. The enzymes that process the HB-EGF transmembrane form are unknown. Accordingly, an in vitro assay was established using a fusion protein in which alkaline phosphatase (AP) replaced the transmembrane and cytoplasmic domains of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agarose beads coated with anti-AP antibodies. Several matrix metalloproteinases (MMPs) were tested for the ability to release soluble HB-EGF in the in vitro system. MMP-3 released soluble 12-kDa immunoreactive and mitogenic HB-EGF within 30 min. On the other hand neither MMP-2 nor MMP-9 had any cleavage activities. A non-cleavable mutant was prepared by replacing the juxtamembrane (JM) region of HB-EGF with the JM region of CD4. The mutant HB-EGF, which in its full-length form was as active a juxtacrine growth factor as was the wild type HB-EGF in vivo, was not cleaved by MMP-3 in the in vitro assay. The C-terminal portion of the cleaved HB-EGF JM-AP that remained attached to the anti-AP beads was N-terminally sequenced and the MMP-3 cleavage site was determined to be Glu151-Asn152, a site within the JM domain. MMP-3 treatment also released soluble HB-EGF in vivo from MC2 cells expressing transmembrane HB-EGF precursor, at a level of about 2-fold above control. It was concluded that MMP-3 cleaves HB-EGF at a specific site in the JM domain and that this enzyme might regulate the conversion of HB-EGF from being a juxtacrine to a paracrine/autocrine growth factor.

Amino Acid Sequence↗
PubMed1997

Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways.

The switch from a quiescent tumor to an invasive tumor is accompanied by the acquisition of angiogenic properties. This phenotypic change likely requires a change in the balance of angiogenic stimulators and angiogenic inhibitors. The nature of the angiogenic switch is not known. Here, we show that introduction of activated H-ras into immortalized endothelial cells is capable of activating the angiogenic switch. Angiogenic switching is accompanied by up-regulation of vascular endothelial growth factor and matrix metalloproteinase (MMP) bioactivity and downregulation of tissue inhibitor of MMP. Furthermore, we show that inhibition of phosphatidylinositol-3-kinase leads to partial inhibition of tumor angiogenesis, thus demonstrating that activated H-ras activates tumor angiogenesis through two distinct pathways. Finally, we show evidence for two forms of tumor dormancy.

Androstadienes↗
PubMed1997

The regulation of neovascularization of matrix metalloproteinases and their inhibitors.

The process of new capillary formation from preexisting vessels, angiogenesis, is a complex physiological event which is strictly controlled, occurring only very rarely under normal conditions. In contrast, there are a number of serious diseases, among them solid tumor growth, rheumatoid arthritis and several eye diseases, which are characterized by unrestricted new capillary growth and which are described as "angiogenic diseases." One of the key events required for successful angiogenesis is extracellular proteolysis. Increased attention has been focused on matrix metalloproteinase (MMP) family of enzymes whose activity is a rate-limiting step in extracellular matrix remodeling. This review will present the accumulating body of evidence, from a number of laboratories, which documents the important role of MMP activity in the regulation of angiogenesis. Taken together, these data suggest that one strategy for controlling the deregulated angiogenesis characteristic of these serious angiogenic diseases may be one which is operative at the level of the control of MMP activity.

Humans↗
PubMed1997

Dysregulated proteolytic balance as the basis of excess extracellular matrix in fibrotic disease.

To investigate mechanisms of tissue fibrosis, we developed a model of ovine fetal bladder fibrosis due to surgically induced obstruction. Tissues were analyzed for matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Active MMP-2 was not detected in obstructed bladders, while latent and active forms were detected in normal bladders. MMP-1 (interstitial collagenase) activity was lower in obstructed bladders. MMP inhibitory activity was increased with obstruction, as were levels of TIMP mRNA and protein. These results indicate that the proteins responsible for collagen degradation are present in the developing bladder, and a shift in the proteolytic balance favoring inhibition of degradation occurs in a model of obstruction-induced fibrosis. This altered proteolytic balance favors accumulation of extracellular matrix and decreased tissue compliance characteristic of this and perhaps other fibrotic conditions.

Animals↗
PubMed1996

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing.

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1-7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4-6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1-6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds.

Animals↗
PubMed1996

Matrix metalloproteinases and their inhibitors in aqueous humor.

Matrix metalloproteinase activity is the rate-limiting step in extracellular matrix degradation. One mechanism by which metalloproteinases are regulated is through the activity of their endogenous inhibitors, the tissue inhibitors of metalloproteinases. Since metalloproteinase activity is a key component of the angiogenic process and many anterior segment structures are largely avascular, we became interested in examining aqueous humor for the presence of metalloproteinases and their endogenous inhibitors. Using zymography, we have identified the presence of several metalloproteinases in normal aqueous humor. Treatment with 4-aminophenylmercuric acetate, an organomercurial which activates latent metalloproteinases, revealed that all metalloproteinases were in their active state. By Western blot analysis, normal aqueous humor was also found to contain at least two tissue inhibitors of metalloproteinases. Subsequent partial purification by two successive chromatographic steps revealed the presence of inhibitory activity against collagenase, endothelial cell DNA synthesis, and angiogenesis on the chick chorioallantoic membrane. The presence of metalloproteinases and their inhibitors in normal aqueous humor, a fluid which bathes avascular ocular structures, suggests that future studies should examine whether an imbalance in this protease/inhibitor family may contribute to the anterior chamber extracellular matrix alterations associated with diseases such as ocular neovascularization and glaucoma.

Animals↗
PubMed1996

Identification of matrix metalloproteinases and metalloproteinase inhibitors in bovine corpora lutea and their variation during the estrous cycle.

Corpora lutea (CL) were collected from cattle to study key physiologic events in angiogenesis. Our objective was to evaluate the activity of matrix metalloproteinases (MMP) and endogenous inhibitors. Corpora lutea were collected 2, 4, 6, 8, 10, 12, 14, and 16 d (n = 3/d) after estrus was first detected. In zymograms, a band of protein migrating at a relative molecular mass (M(r)) of 98 kDa was increased early in the cycle; a M(r) = 88 kDa band was detectable on all days. The molecular weights of these proteins are consistent with the MMP-9 family members. In all samples, a band of enzyme activity was detected at M(r) = 62 kDa, and another band of lesser density was detected at M(r) = 60 kDa. The molecular weights of these proteins are consistent with the MMP-2 family members. An immunoreactive band, detected in all samples with equal density, migrated between M(r) = 27 and 29 kDa, as did the tissue inhibitor of metalloproteinase (TIMP-1) standard. A second band, which was less dense in samples from d 2 through 6, migrated at M(r) = 19 kDa, as did the TIMP-2 standard. A third band was detected in all samples; it migrated at M(r) = 35 kDa, as did the cartilage-derived inhibitor (CDI) standard, and was less dense in d 8 and d 12 through 16 samples. In summary, MMP (gelatinases) and MMP inhibitors are present in developing luteal tissue, and the M(r) = 98 kDa enzyme, CDI, and TIMP-2 varied during the estrous cycle.

Animals↗
PubMed1995

The role of growth factors in vascular cell development and differentiation.

The control of vascular growth and differentiation is a complex system of activity and interaction between positive and negative modulators of these processes. A number of important stimulators and inhibitors of both smooth muscle cells and endothelial cells have now been purified and biochemically characterized. Imbalances in the activity of these factors can result in serious pathologies. In this chapter, we briefly discuss the biology of blood vessel development and growth, review the current literature which describes these stimulators and inhibitors, and discuss current therapeutic strategies designed around these growth modulators.

Animals↗
PubMed1994

Retinoids modulate endothelial cell production of matrix-degrading proteases and tissue inhibitors of metalloproteinases (TIMP).

We have previously reported that vitamin A inhibits the growth of capillary endothelial cells (EC) (Braunhut, S.J., and Palomares, M. (1991) Microvasc. Res. 41, 47-62). In this study, we have analyzed the effect of retinoids on the production and activity of enzymes involved in the proteolytic degradation of extracellular matrix (ECM) by EC. Substrate gel electrophoresis (zymography) revealed several major matrix metalloproteinases (MMP), of approximately 98-96, 72-68, and 46-45 kDa, whose activities were altered in their amounts in the conditioned media (CM) of EC following retinol or retinoic acid treatment when compared to amounts detected in CM of control cells. All of these gelatinases were inactivated by 1,10-phenanthroline, indicating that they were MMPs. MMP inhibitors (MMPI) were also present in these CM and were separated by gel filtration. Four distinct peaks of MMPIs were detected in the CM of EC. Chromatographic profiles indicated that an approximately 27-kDa MMPI was specifically increased in the CM of retinol-treated cells, whereas a 22-18.5-kDa MMPI was increased in CM derived from retinoic acid-treated cells. The MMPI synthesized by retinol-treated EC was immunologically related to tissue inhibitor of metalloproteinases, type 1 (TIMP-1), and the MMPI produced by retinoic acid-treated cells was related to TIMP-2, as indicated by biosynthetic labeling and immunoprecipitation studies as well as Western blot analysis. Therefore, retinol and retinoic acid treatment of EC differentially affects the types and activity of MMPs and MMPIs produced by the cells, distinct changes which are both correlated with growth inhibition.

Animals↗