Non-viral transfer of BDNF and uPA stimulates peripheral nerve regeneration.

Peripheral nerves connect brain and spinal cord with the extremities and inner organs, and nerves injury can lead the disability and social exclusion. Growth factors and other natural stimulators of regeneration processes look very promising as future medicines. In our study, we tested the influence of genetic constructions that contain genes of brain-derived neurotrophic factor and urokinase plasminogen activator on nerve's structure and function after traumatic and ischemic injuries. Injection of pVax1-hBDNF and pVax1-muPA after traumatic injury led to better restoration of nerve's structure and function compared to similar parameters of control group mice. In ischemic injury model pVax1-hBDNF and pVax1-muPA slowed and reduced the damage progression and stimulated nerve regeneration as well. However, the treatment with pVax1-muPA was less effective after the traumatic injury. As we chose a non-viral method of gene delivery during our study the optimal conditions of plasmid intramuscular delivery were also determined.

LDL induces intracellular signalling and cell migration via atypical LDL-binding protein T-cadherin.

Cadherins are a superfamily of adhesion molecules that mediate Ca(2+)-dependent cell-cell adhesion. T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored member of the cadherin superfamily, was initially identified by immunoblotting of vascular cell membranes as an atypical low affinity low density lipoprotein (LDL)-binding protein. It is not known whether this heterophilic interaction is physiologically relevant. Expression of T-cadherin is upregulated in vascular cells during atherosclerosis, restenosis and tumour angiogenesis, conditions characterized by enhanced cell migration and growth. Elevated levels of serum low density lipoproteins (LDL), which result in cholesterol accumulation in vascular wall, is a widely accepted risk factor in atherosclerosis development. Additionally to its metabolic effects, LDL can produce hormone-like effects in a number of cell types. This study has utilized HEK293 cells and L929 cells stably transfected with T-cadherin cDNA to investigate T-cad-dependent responses to LDL. Stable expression of T-cad in both HEK293 and L929 cells results in significantly (p < 0.05) elevated specific surface binding of [I125]-LDL. Compared with mock-transfectants, cells expressing T-cad exhibit significantly (p < 0.01) enhanced LDL-induced mobilization of intracellular Ca(2+)-stores and a significantly (p < 0.01) increased migration toward an LDL gradient (0.1% BSA + 60 microg/ml LDL) in Boyden chamber migration assay. Thus LDL-binding to T-cad is capable of activating physiologically relevant intracellular signaling and functional responses.

LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions.

T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca(2+)-dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [(125)I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca(2+)-dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.

Identification of 130 kDa cell surface LDL-binding protein from smooth muscle cells as a partially processed T-cadherin precursor.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

T-cadherin and signal-transducing molecules co-localize in caveolin-rich membrane domains of vascular smooth muscle cells.

Cadherins are a family of cellular adhesion proteins mediating homotypic cell-cell binding. In contrast to classical cadherins, T-cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This study explores the hypothesis that T-cadherin might function as a signal-transducing protein. Membranes from human and rat vascular smooth muscle cells were fractionated using Triton X-100 solubilization and density gradient centrifugation techniques. We demonstrate that T-cadherin is enriched in a minor detergent-insoluble low-density membrane domain and co-distributes with caveolin, a marker of caveolae. This domain was enriched in other GPI-anchored proteins (CD-59, uPA receptor) and signal-transducing molecules (G alpha s protein and Src-family kinases), but completely excluded cell-cell and cell-matrix adhesion molecules (N-cadherin and beta1-integrin). Coupling of T-cadherin with signalling molecules within caveolae might enable cellular signal transduction.

Characteristics of smooth muscle cell lipoprotein binding proteins (p105/p130) as T-cadherin and regulation by positive and negative growth regulators.

Smooth muscle cells (SMC) express atypical surface low density lipoprotein (LDL) binding proteins of M(r)105 and M(r)130 (p105 and p130) which have been putatively identified as the cell adhesion glycoprotein T-cadherin. Using cultured human and rat aortic SMC and analysis by ligand (LDL)- and immuno-blotting techniques we now confirm identity of p105 and p130 as T-cadherin, as adjudged by sensitivity to PI-PLC cleavage, insensitivity to trypsin degradation in the presence of calcium, and immunoreactivity to anti-T-cadherin peptide antisera. The function of T-cadherin (p105/p130) in the vasculature is unknown. The proteins were downmodulated by the peptide growth factors PDGF-BB, IGF, EGF, and bFGF, but not by vasoactive peptide hormones (angiotensin II, vasopressin, bradykinin, and endothelin). TGF beta, a recognized inhibitor of SMC proliferation, per se had no effect but inhibited growth factor-induced p105/p130 downmodulation. Expression of p105/p130 in quiescent SMC and growth-stimulated SMC (respectively, in serum-free and serum or PDGF-BB containing culture conditions) was increased by forskolin and 8-Br-cyclic GMP, both anti-mitogenic substances, but was unaffected by phorbol ester, calcium ionophores, or calcium antagonists. The findings are compatible with a function for the lipoprotein binding proteins (T-cadherin) in negative regulation of SMC growth.

Identification of an atypical lipoprotein-binding protein from human aortic smooth muscle as T-cadherin.

We have previously described an atypical lipoprotein-binding protein of about 105 kDa (p105) in membranes of vascular smooth muscle cells (VSMCs) that is distinct from currently known lipoprotein receptors. In the present work we have developed a procedure for purification of p105 from human aortic media. Partial sequencing of purified protein has revealed identity of p105 with human T-cadherin. Anti-peptide antisera raised against human T-cadherin recognized a protein spot corresponding to the purified p105 on two-dimensional Western blots. The antisera also inhibited LDL binding to p105 on ligand blots. We conclude that the 105 kDa lipoprotein-binding protein present in human VSMCs is T-cadherin, an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell-cell adhesion proteins.

Ligand selectivity of 105 kDa and 130 kDa lipoprotein-binding proteins in vascular-smooth-muscle-cell membranes is unique.

Using ligand blotting techniques, with low-density lipoprotein (LDL) as ligand, we have previously described the existence of atypical lipoprotein-binding proteins (105 kDa and 130 kDa) in membranes from human aortic medical tissue. The present study demonstrates that these proteins are also present in membranes from cultured human (aortic and mesenteric) and rat (aortic) vascular smooth-muscle cells (VSMCs). To assess the relationship of 105 and 130 kDa lipoprotein-binding proteins to known lipoprotein receptors, ligand binding specificity was studied. We tested effects of substances known to antagonize ligand binding to either the LDL [apolipoprotein B,E (apo B,E)] receptor (dextran sulphate, heparin, pentosan polysulphate, protamine, spermine, histone), the scavenger receptor (dextran sulphate, fucoidin), the very-low-density-lipoprotein (VLDL) receptor [receptor-associated protein (RAP)], or LDL receptor-related protein (RAP, alpha 2-macroglobulin, lipoprotein lipase, exotoxin-A). None of these substances, with the exception of dextran sulphate, influenced binding of LDL to either 105 or 130 kDa proteins. Sodium oleate or oleic acid, known stimuli for the lipoprotein binding activity of the lipolysis-stimulated receptor, were also without effect. LDL binding to 105 and 130 kDa proteins was inhibited by anti-LDL (apo B) antibodies. LDL and VLDL bound to 105 and 130 kDa proteins with similar affinities (approximately 50 micrograms/ml). The unique ligand selectivity of 105 and 130 kDa proteins supports the existence of a novel lipoprotein-binding protein that is distinct from all other currently identified LDL receptor family members. The similar ligand selectivity of 105 and 130 kDa proteins suggests that they may represent variant forms of an atypical lipoprotein-binding protein.

Atypical low density lipoprotein binding site that may mediate lipoprotein-induced signal transduction.

The characteristics of low density lipoprotein (LDL) binding in quiescent cultures of human vascular smooth muscle cells (VSMC) have been further investigated and compared with the characteristics of high affinity LDL binding in human fibroblasts [via the apolipoprotein (apo) B/E receptor] and with the properties of LDL-induced phosphoinositide catabolism in VSMC. In VSMC the bulk of specific 125I-LDL binding occurs at a low affinity site, several characteristics of which are distinct from those of 125I-LDL binding to the apo B/E receptor in fibroblasts. (a) The affinity of LDL binding in VSMC is 25-50 times lower than that in fibroblasts (Kd approximately 50 micrograms/ml versus Kd approximately 2 micrograms/ml). (b) The kinetics of LDL association and dissociation in VSMC are more rapid than those in fibroblasts. (c) In contrast to apo B/E receptor-mediated binding of LDL in fibroblasts, binding of LDL to VSMC is insensitive to heparin, chemical modification of lysine residues, and chelation (with EDTA) of divalent cations. (d) Apo E-free high density lipoprotein 3 displaces labeled LDL more effectively in VSMC than in fibroblasts. (e) The ratio of bound/internalized LDL to degraded LDL differs markedly between fibroblasts and VSMC. LDL-stimulated phosphoinositide catabolism in VSMC, which occurs with an activation constant similar to the Kd for low affinity LDL binding, is insensitive to heparin, modification of lysine and arginine residues in LDL, and chelation of divalent cations. Thus, the atypical low affinity receptor in these cells may mediate the effects of LDL on signal transduction.