Biomedical subjects

Hynda K Kleinman

Publications and source records attributed to Hynda K Kleinman.

At least 19 recordsLinked to original sources

PubMed2006

Neuropeptide Y induces migration, proliferation, and tube formation of endothelial cells bimodally via Y1, Y2, and Y5 receptors.

Previously we discovered that NPY induces ischemic angiogenesis by activating Y2 and Y5 receptors. The receptors that mediate specific steps of the complex process of angiogenesis are unknown. Here, we studied in vitro NPY receptors subtypes involved in migration, proliferation, and differentiation of human endothelial cells. In cells that expressed Y1, Y2, and Y5 receptors, NPY bimodally stimulated migration and proliferation with a 2-fold increase at 10(-12) M and 10(-8) M (high- and low-affinity peaks, respectively). Preincubation of cells with NPY up-regulated the Y5 receptor and markedly enhanced endothelial cell migration and proliferation. NPY-induced endothelial cell migration was mimicked by agonists and fully blocked by antagonists for any specific NPY receptors (Y1, Y2, or Y5), while proliferation was blocked by any two antagonists (Y1+Y2, Y1+Y5, or Y2+Y5), and capillary tube formation on Matrigel was blocked by all three (Y1+Y2+Y5). Thus, NPY-induced angiogenesis requires participation of Y1, Y2, and Y5 receptor subtypes, with the Y5 receptor acting as an enhancer. We propose that these receptors form heteromeric complexes, and the Y1/Y2/Y5 receptor oligomer may be the uncloned Y3 receptor.

Capillaries↗

PubMed2006

Zyxin is upregulated in the nucleus by thymosin beta4 in SiHa cells.

Thymosin beta4 is a 43-amino acid actin-binding protein that promotes cell migration and is important in angiogenesis, wound healing, and tumor metastasis. We searched for genes upregulated by thymosin beta4 and identified zyxin as increased in SiHa cells in the presence of exogenously added thymosin beta4 and when thymosin beta4 is overexpressed using adenoviral vectors. Both zyxin and thymosin beta4 show increased localization in the nucleus. We conclude that thymosin beta4 may exert some of its migration promoting activity via increased zyxin expression.

Active Transport, Cell Nucleus↗

PubMed2006

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair.

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.

Amino Acid Sequence↗

PubMed2006

Angiotropism of human melanoma: studies involving in transit and other cutaneous metastases and the chicken chorioallantoic membrane: implications for extravascular melanoma invasion and metastasis.

Melanoma cell migration along the outside of vessels has been termed "extravascular migratory metastasis" (EVMM), as distinct from intravascular dissemination. Previous studies in both human and experimental melanoma models have shown angiotropism of melanoma cells, suggesting EVMM. Our objectives are to study the mechanism of dissemination of human melanoma cells in the chick chorioallantoic membrane (CAM) and to compare the histopathology in the CAM with that of patients with in transit and other cutaneous melanoma metastases. Human and murine melanoma cells were inoculated onto the CAM and observed over a 10-day period for tumor dissemination. Both human melanoma specimens from 26 patients and melanoma cells growing on the CAM showed the presence of tumor cell angiotropism at the invasive front of the tumor and at some distance from the tumor mass. In addition, a clear progression of melanoma cells spreading on the CAM was observed along the abluminal surface of vessels, where they occupied a perivascular location. By day 10 after injection, small micrometastases had developed along vessels, in a pattern similar to that in transit and other cutaneous melanoma metastases. In addition, the results suggested that the number of micrometastases directly correlated with increasing tumor volume. Taken together, these data suggest that the CAM is a relevant model for studying tumor cell dissemination, and that EVMM may be a mechanism by which some melanoma cells spread to nearby and even distant sites.

Animals↗

PubMed2005

Laminin alpha5 chain metastasis- and angiogenesis-inhibiting peptide blocks fibroblast growth factor 2 activity by binding to the heparan sulfate chains of CD44.

Recently, we reported that the laminin alpha5 synthetic peptide A5G27 (RLVSYNGIIFFLK, residues 2,892-2,904) binds to the CD44 receptor of B16-F10 melanoma cells via the glycosaminoglycans on CD44 and inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner. Here, we have identified the potential mechanism of A5G27 activity using WiDr human colorectal carcinoma cells. WiDr cells bound to the laminin A5G27 peptide via the heparin-like and chondroitin sulfate B glycosaminoglycan side chains of CD44. Cell binding to fibroblast growth factor (FGF2) was blocked by laminin peptide A5G27 but not by either a scrambled version of this peptide or by another laminin peptide known to bind cell surface proteoglycans. FGF2 signaling involving tyrosine phosphorylation was also blocked by laminin peptide A5G27 but was not affected by peptide controls. Finally, we have shown that peptide A5G27 directly blocks FGF2 binding to heparin. Peptide A5G27 has sequence homology to a region on FGF2 that binds heparin and the FGF receptor and is important in FGF2 central cavity formation. We conclude that peptide A5G27 inhibits metastasis and angiogenesis by blocking FGF2 binding to the heparan sulfate side chains of CD44 variant 3, thus decreasing FGF2 bioactivity.

Amino Acid Sequence↗

PubMed2005

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides.

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.

Arginine↗

PubMed2005

Endogenous osteonectin/SPARC/BM-40 expression inhibits MDA-MB-231 breast cancer cell metastasis.

Skeletal metastases occur with high incidence in patients with breast cancer and cause long-term skeletal morbidity. Osteonectin (SPARC, BM-40) is a bone matrix factor that is an in vitro chemoattractant for breast and prostate cancer cells. Increased expression of osteonectin is found in malignant breast tumors. We infected MDA-231 breast cancer cells with an adenovirus expressing osteonectin to examine the role of osteonectin expression in breast cancer cells and its effect on metastasis, in particular to bone. Expression of osteonectin did not affect MDA-231 cell proliferation, apoptosis, migration, cell aggregation, or protease cleavage of collagen IV. However, in vitro invasion of these osteonectin-infected cells through Matrigel and colony formation on Matrigel was decreased. Interestingly, high osteonectin expression in MDA-231 cells inhibited metastasis in a dose-dependent manner to many different organs including bone. The reduction in metastasis may be due to decreased platelet-tumor cell aggregation, because exogenous osteonectin inhibited platelet aggregation in vitro and the high osteonectin expression in MDA-231 cells reduced tumor cell-induced thrombocytopenia in vivo compared with control-infected cells. These studies suggest that high endogenous expression of osteonectin in breast cancer cells may reduce metastasis via reduced invasive activity and reduced tumor cell-platelet aggregation.

Adenoviridae↗

PubMed2005

Bifunctional peptides derived from homologous loop regions in the laminin alpha chain LG4 modules interact with both alpha 2 beta 1 integrin and syndecan-2.

Laminin alpha chains show diverse biological functions in a chain-specific fashion. The laminin G-like modules (LG modules) of the laminin alpha chains consist of a 14-stranded beta-sheet sandwich structure with biologically active sequences found in the connecting loops. Previously, we reported that connecting loop regions between beta-strands E and F in the mouse laminin alpha chain LG4 modules exhibited chain-specific activities. In this study, we focus on the homologous loop regions in human laminin alpha chain LG4 modules using five synthetic peptides (hEF-1-hEF-5). These homologous peptides induced chain-specific cellular responses in various cell types. Next, to examine the dual-receptor recognition model, we synthesized chimeras (cEF13A-cEF13E) derived from peptides hEF-1 and hEF-3. All of the chimeric peptides promoted fibroblast attachment as well as the parental peptides. Attachment of fibroblasts to cEF13A and cEF13B was inhibited by anti-integrin alpha2 and beta1 antibodies and by heparin, while cell adhesion to cEF13C, cEF13D, and cEF13E was blocked only by heparin. Actin organization of fibroblasts on cEF13C was not different from that on hEF-3, but cEF13B induced membrane ruffling at the tips of the actin stress fibers. These results suggest that cEF13B had bifunctional effects on cellular behaviors through alpha2beta1 integrin and heparin/heparan sulfate proteoglycan. We conclude that the approach utilizing chimeric peptides is useful for examining cellular mechanisms in dual-receptor systems.

Actins↗

PubMed2005

Matrix cell adhesion activation by non-adhesion proteins.

Cell adhesion to the extracellular matrix is important in many biological processes. Various ligands and cell surface receptors have been defined. In vitro cell adhesion to matrix proteins and to other 'adhesion' proteins is generally measured on plastic culture substrates. We have found that the presence of low levels of adhesion proteins, e.g. fibronectin, together with high concentrations of non-adhesion proteins, e.g. osteonectin, can promote cell attachment on plastic culture dishes. This promotion of adhesion occurs even when the concentrations of fibronectin, collagen and other adhesive proteins are too low to support cell attachment alone. Other non-adhesive proteins that have similar activity in 'triggering' the attachment of cells to low levels of adhesion molecules include bovine serum albumin (BSA) and cytochrome C. The non-adhesive protein must be added to the plate first, or together with the low amount of the adhesion protein, to 'activate' cell attachment. Adding the adhesion protein fibronectin to the plate first, followed by osteonectin, resulted in no 'activation' of attachment. The non-adhesive protein did not bind to the adhesive protein nor did it alter the level of adhesive protein binding to the substrate. The non-adhesive protein did, however, expose integrin-binding sites of the adhesive protein fibronectin. These data confirm and extend previous data by others demonstrating the role of non-adhesive proteins in regulating the conformation and cell adhesive activity of matrix adhesion proteins on plastic surfaces. Such findings might explain contradictions in the literature about the activity of 'adhesive proteins'.

Animals↗

PubMed2005

Inhibition of VEGF receptors significantly impairs mammary cancer growth in C3(1)/Tag transgenic mice through antiangiogenic and non-antiangiogenic mechanisms.

Cancer growth and progression is often critically influenced by the production of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. VEGF produced by tumor cells stimulates endothelial cell growth through the binding and activation of the KDR/Flk-1 receptor (VEGFR-2) on endothelial cells. Recently, some human breast cancer epithelial cells have been shown to express VEGF receptors, suggesting a potential autocrine-mediated growth stimulation of a subset of cancers by VEGF. We demonstrate that mammary tumors in the C3(1)/Tag transgenic model express VEGF and VEGF receptors and tumor growth is stimulated by this autocrine mechanism. GW654652, an indazolylpyrimidine, is a VEGFRs tyrosine kinase inhibitor that dramatically reduces both angiogenesis and tumor cell growth in this model, as demonstrated using both in vitro and in vivo assays. GW654652 significantly decreased cell proliferation and induced apoptosis in human umbilical vein endothelial cells and M6 mammary tumor cells derived from C3(1)/Tag (Tag: simian virus 40 T-antigen) transgenic mice. A 75% reduction in VEGF-induced angiogenesis was observed with GW654652 using the chick chorioallantoic membrane assay, whereas GW654652 produced an approximately 85% reduction in angiogenesis as assessed by the Matrigel plug assay. A profound inhibitory effect on tumor growth in the C3(1)/Tag transgenic model of human breast cancer was observed with oral administration of GW654652 as measured by delayed tumor onset, decreased multiplicity, reduced tumor volume, and extended animal survival. The antitumor effects of GW654652 were associated with reduced tumor vascularization and no apparent toxicity. Tumor growth, however, rapidly advanced following cessation of treatment. This is the first demonstration that a VEGF receptor inhibitor, GW654652, has a strong inhibitory effect on angiogenesis and tumor progression in a transgenic model of mammary cancer, suggesting that this is a useful approach for preclinical testing of such agents.

Allantois↗

PubMed2005

A simplified laminin nomenclature.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Animals↗

PubMed2005

Thymosin beta4: actin-sequestering protein moonlights to repair injured tissues.

Here, we review the biochemical and molecular properties of thymosin beta(4) (Tbeta(4)), the major actin-sequestering molecule in eukaryotic cells, and its key role in dermal- and corneal-wound healing. Tbeta(4) has several, novel, potential clinical applications in the repair and remodeling of ulcerated tissues and solid organs following hypoxic injuries, such as myocardial infarction and stroke. It might also have important repair functions in the pathophysiologic sequelae that are associated with actin toxicity and with septic shock, such as respiratory distress syndrome, multi-organ failure and severe tissue trauma.

Actins↗

PubMed2005

Matrigel: basement membrane matrix with biological activity.

The basement membrane extracellular matrix contacts epithelial, endothelial, fat and smooth muscle cells. Because this extracellular matrix is so thin, it had been hard to study its composition, structure, and function. An extract of a tumor was found to contain all of the components present in basement and to be very biologically active. This extract, termed Matrigel, Cultrex, or EHS matrix, promotes cell differentiation, and is used to measure the invasive activity of tumor cells. In vivo, it is used for measuring angiogenic inhibitors and stimulators, to improve graft survival, repair damaged tissues, and increase tumor growth.

Animals↗

PubMed2005

Heparanase regulates murine hair growth.

Heparanase is an endoglycosidase that cleaves heparan sulfate, the main polysaccharide component of the extracellular matrix. Heparan sulfate moieties are responsible for the extracellular matrix barrier function, as well as for sequestration of heparin-binding growth factors in the extracellular matrix. Degradation of heparan sulfate by heparanase enables cell movement through extracellular barriers and releases growth factors from extracellular matrix depots, making them bioavailable. Here, we demonstrate a highly coordinated temporospatial pattern of heparanase expression and enzymatic activity during hair follicle cycling. This pattern paralleled the route and timing of follicular stem cell progeny migration and reconstitution of the lower part of the follicle, which is a prerequisite for hair shaft formation. By monitoring in vivo activation of luciferase reporter gene driven by heparanase promoter, we observed activation of heparanase gene transcription at a specific stage of the hair cycle. Heparanase was produced by rat vibrissa bulge keratinocytes, closely related to a follicular stem cell population. Heparanase contributed to the ability of the bulge-derived keratinocytes to migrate through the extracellular matrix barrier in vitro. In heparanase-overexpressing transgenic mice, increased levels of heparanase enhanced active hair growth and enabled faster hair recovery after chemotherapy-induced alopecia. Collectively, our results identify heparanase as an important regulator of hair growth and suggest that cellular mechanisms of its action involve facilitation of follicular stem cell progeny migration and release of extracellular matrix-resident, heparin-bound growth factors, thus regulating hair cycle.

Alopecia↗

PubMed2005

Complex extracellular matrices promote tissue-specific stem cell differentiation.

Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA-developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin-1 or collagen I, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular- and tubular-like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular- and glandular-like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue-stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.

Animals↗

PubMed2004

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides.

Laminins, heterotrimeric glycoproteins in the basement membrane, are involved in diverse biological activities. So far, five alpha, three beta, and three gamma chains have been identified, and at least 15 laminin isoforms exist composed of various combinations of the different three chains. The major cell-surface receptors for laminins are integrins and proteoglycans, such as dystroglycans and syndecans. Previously, we reported that synthetic peptide A4G82 (TLFLAHGRLVFM, mouse laminin alpha4 chain residues 1514-1525) showed strong cell attachment and syndecan binding activities. On the basis of the crystal structure of the LG module and sequence alignment, A4G82 is located in the connecting loop region between beta-strands E and F in the laminin alpha4 chain LG4 module. Here, we have focused on the structural importance of this E-F loop region for the biological activity of the alpha4 chain LG4 module. To determine the importance of the loop structure, we synthesized peptide A4G82X (cyclo-A4G82X, Cys-TLFLAHGRLVFX-Cys, X= norleucine), which was cyclized via disulfide bridges at both the N- and C-termini. The cyclic peptides derived from A4G82X inhibited the heparin binding activity of the alpha4 chain G domain and promoted HT-1080 cell attachment better than the corresponding linear peptides. We determined FLAHGRLVFX as a minimal sequence of cyclo-A4G82X important for cell adhesion and heparin binding using a series of truncated peptides. Moreover, HT-1080 cell attachment to the cyclic peptides was more efficiently blocked by heparin than cell attachment to the linear peptides. Furthermore, the cyclic peptides showed significantly enhanced syndecan-2-mediated cell attachment activity. These results indicate that the activity of A4G82 is highly conformation-dependent, suggesting that the E-F loop structure is crucial for its biological activity.

Amino Acid Sequence↗

PubMed2004

Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo.

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1-92) and N-terminal truncated CCL15(25-92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25-92) is at least 100-fold more potent than CCL15(1-92). Treatment with pertussis toxin (PTX), with anti-CCR1, or with anti-CCR3 antibody inhibits the CCL15(25-92)-induced endothelial cell migration. CCL15(25-92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25-92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.

Animals↗

PubMed2004

Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy.

The laminin alpha5 chain is a component of laminin-10 (alpha5beta1gamma1) and -11 (alpha5beta2gamma1). In this study, we have screened 113 overlapping synthetic peptides from the laminin alpha5 globular domain (G-domain) for cell attachment activity with B16-F10 cells using peptide-coated dishes. Eleven attachment-active peptides were identified. In vivo experimental B16-F10 pulmonary metastasis and primary tumor growth assays found that 4 of the 11 peptides inhibited tumor metastasis and growth and increased apoptosis. These four peptides also blocked tumor cell migration, invasion, and angiogenesis. Two of the peptides were highly homologous and showed significant similarity to sequences in collagens. We sought to identify the B16-F10 cell surface receptors for each of the four active peptides using peptide affinity chromatography. Only one peptide recognized a cell surface protein. Peptide A5G27 (RLVSYNGIIFFLK, residues 2892-2904) bound a diffuse M(r) approximately 120,000-180,000 band that eluted with 2 m NaCl. Glycosidase digestion of the 2 m eluate yielded protein bands of M(r) 90,000 and 60,000 that reacted in Western blot analysis with antibodies to CD44. Immunoprecipitation of the A5G27-bound membrane proteins with various cell surface proteoglycan antibodies confirmed CD44 as the surface receptor for A5G27. Finally, attachment assays to A5G27 in the presence of soluble glycosaminoglycans (GAGs) identified the GAGs of CD44 as the binding sites for A5G27. Our results suggest that A5G27 binds to the CD44 receptor of B16-F10 melanoma cells via the GAGs on CD44 and, thus, inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner.

Amino Acid Sequence↗