D. Stambolsky

Adipose-Derived Stem Cells Stimulate Regeneration of Peripheral Nerves: BDNF Secreted by These Cells Promotes Nerve Healing and Axon Growth De Novo

Transplantation of adipose-derived mesenchymal stem cells (ASCs) induces tissue regeneration by accelerating the growth of blood vessels and nerve. However, mechanisms by which they accelerate the growth of nerve fibers are only partially understood. We used transplantation of ASCs with subcutaneous matrigel implants (well-known in vivo model of angiogenesis) and model of mice limb reinnervation to check the influence of ASC on nerve growth. Here we show that ASCs stimulate the regeneration of nerves in innervated mices limbs and induce axon growth in subcutaneous matrigel implants. To investigate the mechanism of this action we analyzed different properties of these cells and showed that they express numerous genes of neurotrophins and extracellular matrix proteins required for the nerve growth and myelination. Induction of neural differentiation of ASCs enhances production of brain-derived neurotrophic factor (BDNF) as well as ability of these cells to induce nerve fiber growth. BDNF neutralizing antibodies abrogated the stimulatory effects of ASCs on the growth of nerve sprouts. These data suggest that ASCs induce nerve repair and growth via BDNF production. This stimulatory effect can be further enhanced by culturing the cells in neural differentiation medium prior to transplantation.

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 (Gαs protein and Src‐family kinases), but completely excluded cell‐cell and cell‐matrix adhesion molecules (N‐cadherin and β1‐integrin). Coupling of T‐cadherin with signalling molecules within caveolae might enable cellular signal transduction.

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.

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

Cadherins are a superfamily of adhesion molecules that mediate Ca2+-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 Ca2+-stores and a significantly (p < 0.01) increased migration toward an LDL gradient (0.1% BSA + 60 μg/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 Ca2+‐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 [125I]‐LDL that was sensitive to PI‐PLC pre‐treatment of cells. Ca2+‐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.

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 nerves structure and function after traumatic and ischemic injuries. Injection of pVax1-hBDNF and pVax1-muPA after traumatic injury led to better restoration of nerves 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.

Data supporting that adipose-derived mesenchymal stem/stromal cells express angiotensin II receptors in situ and in vitro

This article contains results of analyses of angiotensin II receptors expression in human adipose tissue and stem/stromal cells isolated from adipose tissue. We also provide here data regarding the effect of angiotensin II on intracellular calcium mobilization in adipose tissue derived stem/stromal cells (ADSCs). Discussion of the data can be found in (Sysoeva et al., 2017) [1].