Biomedical subjects

K M Malinda

Publications and source records attributed to K M Malinda.

12 recordsLinked to original sources

PubMed1999

Gp38k, a protein synthesized by vascular smooth muscle cells, stimulates directional migration of human umbilical vein endothelial cells.

Gp38k is a 383-amino-acid secreted glycoprotein expressed by cultured vascular smooth muscle cells during the time of transition from a proliferating monolayer culture to a nonproliferating multilayered (differentiated) culture. Expression continues as the cell culture forms multicellular nodules. Because this transition period involves active cell migration, we evaluated the effects of exogenously added gp38k on vascular endothelial cell (HUVEC) migration and chemotaxis. Here we demonstrate that gp38k acts as a chemoattractant for HUVECs and stimulates cell migration in Boyden chambers at a level comparable to that achieved with the known endothelial cell chemoattractant bFGF. The migration effect is neutralized by the presence of a polyclonal anti-gp38k antibody. Because gp38k expression is also correlated with changes in culture morphology, we also assessed its ability to act as an agonist of HUVEC morphology using cultures growing on Matrigel. We report that gp38k stimulates endothelial cell tubulogenesis in this assay system. These results provide the first evidence that gp38k may function in angiogenesis by stimulating the migration and reorganization of vascular endothelial cells.

Adipokines↗

PubMed1999

Identification of endothelial cell binding sites on the laminin gamma 1 chain.

The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like structures when plated on a laminin-1-rich basement membrane matrix, Matrigel. Laminin-1 is composed of 3 chains, alpha1, beta1, and gamma1. Because laminin-1 is known to contain multiple biologically active sites, we have screened 156 synthetic overlapping peptides spanning the entire laminin gamma1 chain for potential angiogenic sequences. Only 7 of these peptides, designated as C16, C25, C30, C38, C64, C75, and C102, disrupted the formation of capillary-like structures by human umbilical vein endothelial cells on Matrigel. Dose-response experiments in the presence of 50 to 200 microg/mL showed that tube formation was prevented by most peptides at 150 and 200 microg/mL, except for C16, which showed strong activity at all concentrations. Active peptides promoted vessel sprouting from aorta rings and angiogenesis in the chick chorioallantoic membrane assay. In addition, the active peptides also promoted endothelial cell adhesion to dishes coated with 0.1 microg of peptide and inhibited attachment to laminin-1 but not to plastic or fibronectin. Four of the active peptides, C25, C38, C75, and C102, may have cell-type specificity with endothelial cells, since they did not promote PC12 neurite outgrowth or adhesion of B16-F10 melanoma and human submandibular gland cells. These results suggest that specific laminin gamma1-chain peptides have angiogenic activity with potential therapeutic applications.

Allantois↗

PubMed1999

Thymosin beta4 accelerates wound healing.

Angiogenesis is an essential step in the repair process that occurs after injury. In this study, we investigated whether the angiogenic thymic peptide thymosin beta4 (Tbeta4) enhanced wound healing in a rat full thickness wound model. Addition of Tbeta4 topically or intraperitoneally increased reepithelialization by 42% over saline controls at 4 d and by as much as 61% at 7 d post-wounding. Treated wounds also contracted at least 11% more than controls by day 7. Increased collagen deposition and angiogenesis were observed in the treated wounds. We also found that Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay. These results suggest that Tbeta4 is a potent wound healing factor with multiple activities that may be useful in the clinic.

Animals↗

PubMed1999

Identification of laminin alpha1 and beta1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting.

Laminin-1 is a basement membrane glycoprotein that promotes several biological activities including cell attachment, tumor metastasis, and angiogenesis. Angiogenesis plays an important role in tissue formation, reproduction, wound healing, and several pathological conditions. In this study, we screened 405 synthetic peptides from the alpha1 and beta1 chains to identify potential sites on laminin-1 active with endothelial cells. Peptides were initially screened by testing both endothelial cell adhesion to peptide-coated wells and tube formation on Matrigel in the presence of soluble peptide. Twenty active peptides were identified in these screens. A secondary screen using the rat aortic ring sprouting assay identified 13 of the 20 peptides that stimulated endothelial sprouting. Several of these active peptides were also found to stimulate human umbilical vein endothelial cell migration in Boyden chamber assays. Differences in the amount of peptide needed for the response and in the resultant morphologies/responses were observed between the peptides in all of the assays. Our results suggest that several active domains on laminin-1 may play important roles in stimulating different steps in angiogenesis.

Amino Acid Sequence↗

PubMed1998

Cell binding sequences in mouse laminin alpha1 chain.

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.

Amino Acid Sequence↗

PubMed1998

Thymosin alpha 1 stimulates endothelial cell migration, angiogenesis, and wound healing.

In wound healing, lymphoid cells release soluble factors that attract fibroblasts and macrophages, initiating repair, endothelial cell migration, angiogenesis, and matrix production. We analyzed the effect of thymosin alpha1 (Talpha1) on endothelial cell migration, angiogenesis, and wound healing. Talpha1, a 28 amino acid peptide initially isolated from the thymus, enhanced the morphologic differentiation of endothelial cells and was a potent chemoattractant for endothelial cells and monocytes in vitro. In vivo, Talpha1 stimulated angiogenesis in a subcutaneous model. When given either topically or i.p., it accelerated wound healing in a punch model, demonstrating that Talpha1 promotes angiogenesis and wound healing.

Adjuvants, Immunologic↗

PubMed1998

Differential effects of hepatocyte growth factor isoforms on epithelial and endothelial tubulogenesis.

Hepatocyte growth factor (HGF)/scatter factor (SF) is a pleiotropic cytokine that acts as a mitogen, motogen, and morphogen for a variety of cell types. HGF/NK1 and HGF/NK2 are two naturally occurring truncated variants of HGF/SF, which extend from the NH2 terminus through the first and second kringle domain, respectively. Although these variants have been reported to have agonistic or antagonistic activity relative to HGF/SF in assays of cell proliferation and motility, their potential morphogenic activity has not been investigated. To address this issue, we assessed the ability of HGF/NK1 and HGF/NK2 to induce tube formation by (a) MCF-10A mammary epithelial cells grown within collagen gels and (b) human umbilical vein endothelial (HUVE) cells grown on Matrigel. We found that HGF/NK1 stimulated tubulogenesis by both MCF-10A and HUVE cells, whereas HGF/NK2 did not stimulate tubulogenesis, but efficiently antagonized the morphogenic effect of full-length HGF/SF. HGF/NK1 and HGF/NK2 also had agonistic and antagonistic effects, respectively, on MCF-10A cell proliferation and HUVE cell migration. These results demonstrate that HGF/NK1, which only consists of the NH2-terminal hairpin and first kringle domain, is sufficient to activate the intracellular signaling pathways required to induce morphogenic responses in epithelial and endothelial cells. In contrast, HGF/NK2, which differs from HGF/ NK1 by the presence of the second kringle domain, is devoid of intrinsic activity but opposes the effects of HGF/SF. The differential properties of the two HGF/SF isoforms provide a basis for the design of more potent HGF/SF agonists and antagonists.

Cell Division↗

PubMed1997

Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells.

Thymosin beta 4 (T beta 4) is a 4.9 kDa polypeptide that interacts with G-actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. T beta 4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of T beta 4 in endothelial cell migration. Our results demonstrate that T beta 4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four- to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to T beta 4 treatment, suggesting that the migration activity of T beta 4 was endothelial cell-specific. T beta 4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. T beta 4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that T beta 4 stimulated cell migration in vivo. These results provide the first direct evidence that T beta 4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.

Cell Movement↗

PubMed1996

The laminins.

The laminins are a family of extracellular matrix glycoproteins localized in the basement membrane that separates epithelial cells from the underlying stroma. They are also found in basement membrane surrounding fat, muscle and peripheral nerve cells. The laminins are large trimeric glycoproteins comprising three disulphide-bonded chains. They are the first extracellular matrix molecules to be observed in the developing embryo and have potent biological activities. In addition, there have been new developments in the number and localization of the homologues of the laminin chains and the role of laminin in neuromuscular disease. Their primary role is in cell-matrix attachment, but many additional biological activities, including promoting cell growth and migration, tumour growth and metastases, neurite outgrowth, nerve regeneration, wound repair and graft survival, have been demonstrated. Many of these biological activities are duplicated by proteolytic fragments of laminin and by small laminin-derived synthetic peptides. These laminin-derived peptides may be useful clinical reagents for accelerating wound healing with minimal scarring or for blocking tumour metastases.

Animals↗

PubMed1995

Four-dimensional microscopic analysis of the filopodial behavior of primary mesenchyme cells during gastrulation in the sea urchin embryo.

During gastrulation of the sea urchin embryo, primary mesenchyme cells (PMCs) migrate from the vegetal pole to a site below the equator of the embryo where they form a ring-like structure and begin producing the larval skeleton. As these cells migrate, they extend and retract filopodia which appear to interact with the basal lamina and underlying ectoderm. To better characterize this behavior in vivo, we studied PMC migration using differential interference contrast (DIC) microscopy in combination with four-dimensional imaging (x, y, z space and time). We were able to determine the persistence and direction of extension of each filopodium and were also able to observe the dynamic behavior of each using colorized movie loops. This analysis showed that: (1) Most filopodia are quite transient, usually persisting for less than 0.5-6.0 min, during which time they continuously survey their surroundings; (2) PMCs extend an average of 121 filopodia/hr during migration; (3) the initial direction of extension of filopodia from the cell body is random, with just as many filopodia projecting toward as away from the direction of migration; (4) as a consequence of (2) and (3) above, each PMC explores the area surrounding its cell body approximately once every 5 min; (5) PMCs nearer to the target site migrate faster than those located farther away. To further investigate filopodial distribution, confocal microscopy was used to collect z series of PMCs transplanted to different locations in the embryo and fixed during migration. We found that more filopodia tended to be distributed toward the target site as cells approached the ring, suggesting that filopodial distribution may reflect regional differences in directional cues.

Animals↗

PubMed1994

Primary mesenchyme cell migration in the sea urchin embryo: distribution of directional cues.

The directional migration of the primary mesenchyme cells (PMCs) of the sea urchin embryo is a critical step in the process of gastrulation. Although interactions between the migrating cells and the blastocoel environment are necessary for guiding the PMCs to their subequatorial target site, the nature of these interactions and the localization of guidance cues involved in directing the cells are not yet known. Previous studies have suggested that PMC migration is the result of random exploration and selective trapping at the target site by a pattern of adhesiveness in the ectoderm or basal lamina. To better characterize the distribution of guidance cues in the blastocoel we used a combination of time-lapse microscopy, microsurgery, and fluorescence photoablation to study the behavior of the migrating cells. By using fluorescence time-lapse microscopy, and a two-dimensional random-walk analysis of cell trajectories, we demonstrated that fluorescently labeled PMCs injected near the animal pole move in a directed fashion over a relatively long distance to reach the target site. This suggests that guidance cues are distributed globally throughout the embryo and are not restricted to the immediate ring area. To further test this hypothesis we investigated the migratory behavior of PMCs that were prevented from interacting directly with the target site. First, we examined the behavior of PMCs injected into animal embryo fragments lacking the target site. We found that PMCs move to the vegetal-most area of such embryo fragments, regardless of their size. Second, we studied the effects of photoablating a stripe of ectoderm between PMCs injected at the animal pole region (APR) and the target site. PMCs were found to accumulate along the ablated stripe and were unable to cross it for up to 6 hr after ablation. We also examined the migratory behavior of endogenous PMCs in embryos treated with lithium, a vegetalizing agent which shifts the position of the PMC ring toward the animal pole. We found that PMCs accumulated along an ablated stripe of ectoderm positioned below the shifted target site, suggesting that endogenous PMCs follow a set of directional cues to the target site which may be similar to those used by PMCs injected into the APR. As a whole, these results suggest that migrating PMCs follow a set of directional cues that are widely distributed throughout the blastocoel and that may be arranged in a gradient.

Animals↗

PubMed1993

Size regulation and morphogenesis: a cellular analysis of skeletogenesis in the sea urchin embryo.

The formation of the skeleton is a central event in sea urchin morphogenesis. The skeleton serves as a framework for the larval body and is the primary determinant of its shape. Previous studies have shown that the size of the skeleton is invariant despite wide experimentally induced variations in the number of skeleton-forming primary mesenchyme cells (PMCs). In the present study, we have used PMC transplantation, fluorescent cell markers and confocal laser scanning microscopy to analyze cellular aspects of skeletal patterning. Labeling of embryos with 5-bromodeoxyuridine demonstrates that the entire embryonic phase of skeletal morphogenesis occurs in the absence of PMC division. During embryogenesis, skeletal rods elongate by one of two mechanisms; either preceded by a cluster (plug) of PMCs or by extending along an existing PMC filopodial cable. Elongation of skeletal rods occurs exclusively by the addition of new material at the rod tips, although radial growth (increase in rod thickness) occurs along the length of the rods. Photoablation of a distinctive region of ectoderm cells at the arm tip results in an inhibition of skeletal rod elongation, indicating that a local ectoderm-PMC interaction is required for skeletal growth. The regulation of skeletal patterning was also examined in embryos that had been microinjected with additional PMCs and in half-sized larvae derived from blastomeres isolated at the 2-cell stage. Microinjection of 50-100 PMCs into the blastocoel at the mesenchyme blastula stage leads to an increase in the numbers of PMCs along all skeletal rods and a two-fold increase in the number of cells in the plugs, yet no increase in the length of the skeletal rods. The length of the anal rods can, however, be increased by microinjecting developmentally 'young' PMCs into the arm tips of late stage embryos. We find that the rate of skeletal rod elongation is independent of both the mode of rod growth (chain or plug) and the number of PMCs in the plug at the growing rod tip. Instead, the rate of elongation appears to be strictly regulated by the quantity of ectodermal tissue present in the embryo. These studies provide new information concerning normal mechanisms of skeletal growth and patterning and lead us to propose a model for the regulation of skeleton size based upon an intrinsic PMC 'clock' and an ectoderm-derived signal that regulates the rate of skeletal rod elongation.

Animals↗