Darwin Eton

Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

Cell surface receptors play major roles in the mobilization and homing of progenitor cells from the bone marrow to peripheral tissues. CXCR4 is an important receptor that regulates homing of leucocytes and endothelial progenitors in response to the chemokine stromal cell‐derived factor‐1 (SDF‐1). Ionic calcium is also known to regulate chemotaxis of selective bone marrow cells (BMCs) through the calcium‐sensing receptor, CaR. Here we show that calcium regulates CXCR4 expression and BMC responses to SDF‐1. CaCl2 treatment of BMC induced a time‐ and dose‐dependent increase in both the transcription and cell surface expression of CXCR4. BMC subpopulations expressing VEGFR2+, CD34+ and cKit+/Sca‐1+ were especially sensitive to calcium. The effects were blocked by calcium influx inhibitors, anti‐CaR antibody and the protein synthesis inhibitor cycloheximide, but not by the CXCR4 antagonist AMD3100. Calcium treatment also enhanced SDF‐1‐mediated CXCR4 internalization. These changes were reflected in significantly improved chemotaxis by SDF‐1, which was abolished by AMD3100 and by antibody against CXCR4. Calcium pre‐treatment improved homing of CD34+ BMCs to ischemic muscle in vivo, and enhanced revascularization in ischemic mouse hindlimbs. Our results identify calcium as a positive regulator of CXCR4 expression that promotes stem cell mobilization, homing and therapy.

Smooth muscle cells improve endothelial cell retention on polytetrafluoroethylene grafts in vivo

OBJECTIVE We investigated the influence of smooth muscle cells (SMC) on endothelial cell (EC) retention on polytetrafluoroethylene (PTFE) grafts and the effect of SMC seeding on intimal hyperplasia in vivo in a rabbit model. METHODS Fibronectin-coated PTFE grafts (4 mm diameter) were seeded with either EC alone, SMC alone, or SMC followed 24 hours later by EC. The grafts were connected to an extracorporal aortic shunt for 1 hour or were individually implanted for 1, 30, and 100 days into the infrarenal aorta as an end-to-side bypass graft. The number of retained cells was compared at 1 hour and at 1 day after implantation. Neointimal thickness was measured 30 and 100 days after implantation. RESULTS After 1-hour exposure to blood flow, EC retention rate was greater (P <.005) if seeded on top of SMC (98% +/- 2%; n = 8) versus being seeded alone (65 +/- 11%; n = 8). SMC retention rate was 95 +/- 5% (n = 8) when seeded alone. Similar cell retention was obtained 1 day after implantation. After 30-day implantation the neointima was thicker in grafts seeded with EC and SMC (282 +/- 136 microm; n = 3) than with EC only (52 +/- 45 microm; n = 3; P <.001). However, the neointimal thickness for dual-cell-seeded grafts (126 +/- 60 microm; n = 3) was not significantly different (P =.09) from EC-seeded grafts (79 +/- 48 microm; n = 3) after 100-day implantation. CONCLUSION EC retention on PTFE grafts in vivo is improved if seeded over a layer of SMC. Further studies are needed to determine whether overlying EC modulate proliferation of underlying SMC.

Enhanced cell therapy strategy to treat chronic limb-threatening ischemia

Intermittent programmed compression of the chronically ischemic limb is associated with arteriogenesis. However, progenitor cell elements contributing to this neovascularization are typically diminished in number and function in the elderly dysvascular patient, particularly in the presence of diabetes, renal insufficiency, and cardiac disease. Granulocyte-colony stimulation factor (G-CSF) dramatically boosts the circulating progenitor cell count. G-CSF was administered in 2 patients being treated for ischemic wounds with an intermittent programmed pneumatic compression device (PPCD). Both had comorbidities associated with diminished circulating progenitor cell counts. Remarkable clinical, hemodynamic, and angiographic improvement was observed. Further study of this synergistic strategy is warranted.

Injury induced neointima formation and its inhibition by retrovirus-mediated transfer of nitride oxide synthase gene in an in-vitro human saphenous vein culture model

Human saphenous veins were cultured to characterize neointima formation and feasibility of gene transfer to inhibit the intimal proliferative response to injury. Mechanical injury was introduced by abrading the luminal surface of the vein patch with a sterile cotton bud. Both injured and non-injured vein patches were cultured and transduced with retroviral vectors carrying marker or therapeutic genes. After a 14-day culture, the thickness of the intimal layer of non-injured vein patches reached 90+/-28 microm at the edge and 61+/-22 microm at the center (n=29) from the original 22+/-12 microm at harvest (n=6, P=0.02). Mechanical injury to the intimal surface prior to culture resulted in an exaggerated proliferative response. The intimal thickness of injured vein patches increased from 3.4+/-1 microm right after injury to 128+/-23 microm (n=12, P<0.001) at the edge after 14-day culture. Genes were transduced efficiently into a luminal layer of cultured veins using a pseudotyped murine leukemia viral vector. Transduction of gene encoding nitric oxide synthase resulted in reduction of neointima formation to 33+/-7 microm (n=12) at the edge after 14-day culture compared to 90 microm (P<0.01) seen in untransduced non-injured vein patches. Marker gene transduction did not alter intimal proliferative response or its immunohistochemical profile. The data suggest that cultured vein can be used as a model for studying the effects of injury to blood vessels and to evaluate the effects of candidate therapeutic genes.

Blood-derived smooth muscle cells as a target for gene delivery

OBJECTIVE To examine the feasibility of using blood-derived smooth muscle cells (BD-SMCs) as a target for to deliver therapeutic proteins. MATERIALS AND METHODS Mononuclear cells (MNC) were isolated from peripheral blood. The outgrowth colonies from MNC culture were differentiated into BD-SMCs in media containing platelet-derived growth factor BB. Phenotypic characterization of BD-SMCs was assessed by immunocytochemistry. Cell proliferation, gene transfer efficiency with a retroviral vector, apoptosis, and the biological activity of the transduced gene product from the BD-SMCs were evaluated in vitro and in vivo in comparison with vascular derived SMC (VSMCs). RESULTS BD-SMCs stained positive for SMC markers. No significant difference was observed between BD-SMCs and VSMCs in cell proliferation, migration, adhesiveness, and gene transfer efficiency. After BD-SMCs were transduced with a retroviral vector carrying the secreted alkaline phosphatase gene (SEAP), 174 +/- 50 mug biologically active SEAP was produced per 10(6) cells over 24 hours. After injecting 5 x 10(6) cells expressing SEAP intravenously into rabbits, SEAP concentration increased significantly in the circulation from 0.14 +/- 0.04 mug/ml to 2.34 +/- 0.16 mug/ml 3 days after cell injection (P < .01, n = 3). Circulating levels of SEAP decreased to 1.76 mug /ml 1 week later and remained at this level up to 8 weeks, then declined to pre-cell injection level at 12 weeks. VSMC in vivo gene expression data were equivalent. CONCLUSION BD-SMCs have similar characteristics to mature VSMCs and can be used as a novel target for gene transfer to deliver a therapeutic protein.

71 – Endovascular Research: Stents

The need to perform vessel reconstruction without disruption of tissue planes has led to a variety of innovative alternatives to conventional open surgical bypass procedures. The biological reaction to endoprosthesis is a phenomenon that occurs when host tissue responds to the presence of foreign material. The characteristics of endoprosthetic biomaterials determine the nature and extent of immediate and long-term responses. In stents, a thin layer of metal oxide, which varies depending on the composition of the material and industrial processing used in the surface finishing, provides the ultimate interface between the stent and host tissue. Examples of such materials are evident in the Palmaz stent and Z stent, and in numerous inferior vena cava filters, which are composed of stainless steel; nitinol is used in Wallstents and Mediloy is used in the Medivent stent. As technology improves, so must the necessary standardization of research techniques used to evaluate biocompatibility, tissue reaction, and patency following the procedure. Any new clinical intervention must first be thoroughly investigated at the laboratory level. Flow models may be employed to examine the feasibility of a new technique, followed by animal models to assess safety and clinical trials to help evaluate the efficacy for widespread use.