Monocyte Derivatives Promote Angiogenesis and Myocyte Survival in a Model of Myocardial Infarction

In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-α, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling.

ENDOTHELIAL NO-SYNTHASE GENE TRANSFER RESTORES REGENERATIVE CAPACITY OF ENDOTHELIAL PROGENITOR CELLS FROM PATIENTS WITH CORONARY ARTERY DISEASE

Background: Endothelial progenitor cells (EPCs) from patients with coronary arterydisease (CAD) or CAD risk factors exhibit greatly reduced regenerativecapacity, which likely contributes to the relatively modest nature of the benefit seen in recent clinical trials of autologous cell therapy postmyocardial infarction. We hypothesized that eNOS overexpression will improve the functional capacity of EPCs from these patients. Methods and Results: EPCs were isolated from the peripheral blood ofpatients with high Framingham risk scores (FRS > 15%) and were transducedusing lentiviral vectors containing either eNOS or GFP (sham). We observed that eNOS-transduction significantly improved migration toward chemotactic factors(VEGF and SDF-1) compared to sham-transduced cells. EPCs were co-cultured witha mature endothelial cell (EC) line on Matrigel to measure their pro-angiogenicfunction in vitro. eNOS-transduced EPCs induced longer angiogenic tubes withmore branch points compared to sham-transduced cells, and exhibited higherassociation with EC tubes. We did not observe a significant difference in the adhesion of EPCs to an EC layer pre-activated with TNF-?, suggesting that the association to angiogenictubes is likely through a different mechanism. In immunodeficient mice, eNOS-transduced EPCs resulted in significant improvement in ischemic hindlimbperfusion compared to sham-transduced cells. PCR arrays revealed changes in angiogenic and pro-survivalgene expression in response to eNOS overexpression, providing preliminaryinsight into the mechanisms underlying its beneficial actions. Conclusions: The present data show that reduced regenerative activity of EPCsisolated from CAD patients can be significantly improved by the overexpressionof eNOS. The combination of cell and gene therapy may improve the efficacy ofautologous cell therapies for cardiovascular disease.

Interleukin-6 Stimulates Circulating Blood-Derived Endothelial Progenitor Cell Angiogenesis in vitro

Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G > C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells ( P <0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner ( P <0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects ( P <0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.

Bone Marrow-Derived Aldehyde Dehdrogenase Expressing Cells Possess Endothelial Progenitor Function in Addition to Hematopoietic Repopulating Ability and Aid in Blood Flow Recovery after Acute Ischemic Injury.

Human bone marrow (BM) is a heterogeneous compartment of stem and progenitor cells from hematopoietic, endothelial, and mesenchymal lineages. These cell types may be recruited to sites of new blood vessel formation including ischemic tissues and tumor microenvironments. Prospective isolation of cells with neoangiogenic function has proven difficult due to the lack of surface markers specific to endothelial precursors. We have previously characterized a novel population of reconstituting hematopoietic stem and progenitor cells from human umbilical cord blood by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity (Hess, Blood 2004). In the current study, we isolated ALDHhi and ALDHlo cells with low side scatter properties from human BM, and assayed for hematopoietic and angiogenic function. The ALDHhi subset represented 0.8±0.2% of total nucleated BM cells whereas ALDHlo cells were 10-fold more abundant (8.2±1.3%, n=5). ALDHhi cells highly expressed cell surface molecules associated with hematopoietic (89.1±1.3% CD45+, 70.5±7.5% CD117+) and endothelial progenitor function (76.5±1.8 CD34+, 72.0±7.6% CD133+). ALDHhi cells also expressed mature macrophage/monocyte markers (24.2±3.9% CD11b+), whereas mature lymphocyte markers (3.5±0.5% CD3+, 5.1±0.3% CD19+), and mature endothelial cell markers (2.6±0.8% VEGFR2+) were rare. The corresponding ALDHlo population demonstrated a significantly reduced frequency of cells expressing a primitive phenotype (8.6±1.5% CD34+, 1.4±0.4% CD117+, 2.2±0.6% CD133+, p&lt;0.05), and a concurrent increase in contaminating lymphocytes (19.8±3.6% CD3+, 10.8±1.0% CD19+, p&lt;0.05). Injection of 104-2x105 BM-derived ALDHhi cells consistently produced hematopoietic repopulation in sublethally irradiated NOD/SCID (n=9) and NOD/SCID beta-2 microglobulin (B2M) null mice (n=13). Equivalent doses of ALDHlo cells produced no human engraftment. Culture of ALDHhi cells in endothelial growth media supplemented with angiogenic growth factors (VEGF, hFGF, IGF, EGF) produced colonies of cells that expressed CD34 and the mature endothelial cell marker CD144 (VE-cadherin). To test the neoangiogenic function of ALDHhi cells in vivo, we performed femoral artery ligation in the hind limb of NOD/SCID B2M null mice, and transplanted purified ALDHhi or ALDHlo cells via the tail vein within 24 hours of ischemic injury. The recovery from acute ischemic injury was calculated by the ratio of blood flow in the ischemic versus the non-ischemic leg by laser Doppler perfusion imaging, performed twice weekly over 28 days. In comparison to PBS-injected control mice (n=6) and mice transplanted with 5x105 ALDHlo cells (n=6), mice transplanted with 105 ALDHhi cells (n=7) showed enhanced blood flow to the ischemic limb by day 7 post-injection (p&lt;0.05). This enhanced recovery of blood flow was maintained throughout the 28-day monitoring period. We are currently investigating the phenotype(s) of human cells recruited to the ischemic area and implicated in the regeneration of vascular function. In summary, the ALDHhi population from human BM is functionally heterogeneous and contains cells with both hematopoietic and endothelial activities. These findings may have implications in the development of cellular therapies for the treatment of patients with acute vascular injury or peripheral vascular disease.

Macrophage colony stimulating factor modulates the development of hematopoiesis by stimulating the differentiation of endothelial cells in the AGM region

Definitive hematopoietic stem cells arise in the aorta–gonad–mesonephros (AGM) region from hemangioblasts, common precursors for hematopoietic and endothelial cells. Previously, we showed that multipotential hematopoietic progenitors and endothelial cells were massively produced in primary culture of the AGM region in the presence of oncostatin M. Here we describe a role for macrophage–colony-stimulating factor (M-CSF) in the development of hematopoietic and endothelial cells in AGM culture. The number of hematopoietic progenitors including multipotential cells was significantly increased in the AGM culture of op/opembryos. The addition of M-CSF to op/op AGM culture decreased colony-forming unit (CFU)-GEMM, granulocyte macrophage–CFU, and erythroid–CFU, but it increased CFU-M. On the other hand, the number of cells expressing endothelial markers, vascular endothelial-cadherin, intercellular adhesion molecule 2, and Flk-1 was reduced in op/op AGM culture. The M-CSF receptor was expressed in PCLP1+CD45− cells, the precursors of endothelial cells, and M-CSF up-regulated the expression of more mature endothelial cell markers—VCAM-1, PECAM-1, and E-selectin—in PCLP1+CD45− cells. These results suggest that M-CSF modulates the development of hematopoiesis by stimulating the differentiation of PCLP-1+CD45− cells to endothelial cells in the AGM region.