Kwijun Park

Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration.

Objective— We demonstrated previously that mouse embryonic stem (ES) cell–derived vascular endothelial growth factor receptor-2 (VEGF-R2)–positive cells can differentiate into both vascular endothelial cells and mural cells. This time, we investigated kinetics of differentiation of human ES cells to vascular cells and examined their potential as a source for vascular regeneration. Methods and Results— Unlike mouse ES cells, undifferentiated human ES cells already expressed VEGF-R2, but after differentiation, a VEGF-R2-positive but tumor rejection antigen 1-60 (TRA1-60)–negative population emerged. These VEGF-R2-positive but tumor rejection antigen 1-60–negative cells were also positive for platelet-derived growth factor receptor &agr; and &bgr; chains and could be effectively differentiated into both VE-cadherin+ endothelial cell and &agr;-smooth muscle actin+ mural cell. VE-cadherin+ cells, which were also CD34+ and VEGF-R2+ and thought to be endothelial cells in the early differentiation stage, could be expanded while maintaining their maturity. Their transplantation to the hindlimb ischemia model of immunodeficient mice contributed to the construction of new blood vessels and improved blood flow. Conclusions— We could identify the differentiation process from human ES cells to vascular cell components and demonstrate that expansion and transplantation of vascular cells at the appropriate differentiation stage may constitute a novel strategy for vascular regenerative medicine.

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

Background We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration. Methods and Results After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity. Conclusions Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.

Different Differentiation Kinetics of Vascular Progenitor Cells in Primate and Mouse Embryonic Stem Cells

Background—We demonstrated that vascular endothelial growth factor receptor 2 (VEGF-R2)-positive cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells and mural cells to suffice as vascular progenitor cells (VPCs). Here we examined whether VPCs occur in primate ES cells and investigated the differences in VPC differentiation kinetics between primate and mouse ES cells. Methods and Results—In contrast to mouse ES cells, undifferentiated monkey ES cells expressed VEGF-R2. By culturing these undifferentiated ES cells for 4 days on OP9 feeder layer, VEGF-R2 expression disappeared, and then reappeared after 8 days of differentiation. We then isolated these VEGF-R2–positive and vascular endothelial cadherin (VEcadherin)-negative cells by flow cytometry sorting. Additional 5-day reculture of these VEGF-R2+ VEcadherin− cells on OP9 feeder layer resulted in the appearance of platelet endothelial cell adhesion molecule-1 (PECAM1)-positive, VEcadherin-positive, endothelial nitric oxide synthase (eNOS)-positive endothelial cells. On a collagen IV-coated dish in the presence of serum, these cells differentiated into smooth muscle actin (SMA)-positive and calponin-positive mural cells (pericytes or vascular smooth muscle cells). Addition of 50ng/mL VEGF to the culture on a collagen IV-coated dish resulted in the appearance of PECAM1+ cells surrounded by SMA+ cells. In addition, these differentiated VEGF-R2+ cells can form tube-like structures in a 3-dimensional culture. Conclusion—Our findings indicate that differentiation kinetics of VPCs derived from primate and mouse ES cells were different. Differentiated VEGF-R2+ VEcadherin− cells can act as VPCs in primates. To seek the clinical potential of VPCs for vascular regeneration, investigations of primate ES cells are indispensable.

Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3‐kinase (PI3K). AM promoted re‐endothelialization in vitro of wounded monolayer of HUVECs and neo‐vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA‐ and PI3K‐dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.

Transplantation of vascular cells derived from human embryonic stem cells contributes to vascular regeneration after stroke in mice.

BackgroundWe previously demonstrated that vascular endothelial growth factor receptor type 2 (VEGF-R2)-positive cells induced from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) and these vascular cells construct blood vessel structures in vitro. Recently, we have also established a method for the large-scale expansion of ECs and MCs derived from human ES cells. We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain.MethodsPhosphate buffered saline, human peripheral blood mononuclear cells (hMNCs), ECs-, MCs-, or the mixture of ECs and MCs derived from human ES cells were intra-arterially transplanted into mice after transient middle cerebral artery occlusion (MCAo).ResultsTransplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes. The cerebral blood flow and the vascular density in the ischemic striatum on day 28 after MCAo had significantly improved in ECs-, MCs- and ECs+MCs-transplanted mice compared to that of mice injected with saline or transplanted with hMNCs. Moreover, compared to saline-injected or hMNC-transplanted mice, significant reduction of the infarct volume and of apoptosis as well as acceleration of neurological recovery were observed on day 28 after MCAo in the cell mixture-transplanted mice.ConclusionTransplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.

The role of mineralocorticoid receptor expression in brain remodeling after cerebral ischemia.

Mineralocorticoid receptors (MRs) are classically known to be expressed in the distal collecting duct of the kidney. Recently it was reported that MR is identified in the heart and vasculature. Although MR expression is also found in the brain, it is restricted to the hippocampus and cerebral cortex under normal condition, and the role played by MRs in brain remodeling after cerebral ischemia remains unclear. In the present study, we used the mouse 20-min middle cerebral artery occlusion model to examine the time course of MR expression and activity in the ischemic brain. We found that MR-positive cells remarkably increased in the ischemic striatum, in which MR expression is not observed under normal conditions, during the acute and, especially, subacute phases after stroke and that the majority of MR-expressing cells were astrocytes that migrated to the ischemic core. Treatment with the MR antagonist spironolactone markedly suppressed superoxide production within the infarct area during this period. Quantitative real-time RT-PCR revealed that spironolactone stimulated the expression of neuroprotective or angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), whereas immunohistochemical analysis showed astrocytes to be cells expressing bFGF and VEGF. Thereby the incidence of apoptosis was reduced. The up-regulated bFGF and VEGF expression also appeared to promote endogenous angiogenesis and blood flow within the infarct area and to increase the number of neuroblasts migrating toward the ischemic striatum. By these beneficial effects, the infarct volume was significantly reduced in spironolactone-treated mice. Spironolactone may thus provide therapeutic neuroprotective effects in the ischemic brain after stroke.

Inhibition of hepatic damage and liver fibrosis by brain natriuretic peptide

Anti‐fibrotic and organ protective effects of brain natriuretic peptide (BNP) have been reported. In this study, effects of BNP on liver fibrosis were examined in the carbon tetrachloride (CCl4)‐induced liver fibrosis model using BNP‐transgenic (Tg) and wild‐type (WT) mice. Twice‐a‐week intraperitoneal injections of CCl4 for 8 weeks resulted in massive liver fibrosis, augmented transforming growth factor (TGF)‐β1 and type I procollagen α1 chain (Col1a1) mRNA expression, and the hepatic stellate cell (HSC) activation in WT mice, all of which were significantly suppressed in Tg mice. These observations indicate that BNP inhibits liver fibrosis by attenuating the activation of HSCs.