Prostacyclin-producing human mesenchymal cells target H19 lncRNA to augment endogenous progenitor function in hindlimb ischaemia.

Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells.

Osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents(1,2). In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT(3).

Targeted delivery of carbaprostacyclin to ischemic hindlimbs enhances adaptive remodeling of the microvascular network.

Prostacyclin and its stable analogs play an important vascular protective role by promoting angiogenesis, but their role in arteriolar growth is unclear. Here, we examined the effect of prostacyclin stable analog carbaprostacyclin on arteriolar growth in mouse hindlimb ischemia. Using an osmotic-controlled release system to continuously deliver carbaprostacyclin or saline (control) to ischemic mouse hindlimbs for up to 14 days, we found that blood perfusion was significantly better at 7 and 14 days in carbaprostacyclin-treated mice than in saline-treated mice. Microscopic examination of the microvasculature showed more morphological signs of arteriolar formation in carbaprostacyclin- versus saline-treated legs. A double-blind, quantitative microcomputed tomography analysis indicated that carbaprostacyclin-treated legs had markedly increased vascular volume and small- to medium-sized vessel numbers that correspond to decreased vessel separation. A proteome profiler antibody array demonstrated that carbaprostacyclin-treated ischemic muscles secreted significantly higher amounts of acidic fibroblast growth factor and other chemokines. Conditioned media containing those secreted factors promoted smooth muscle cell growth and migration. Additionally, increased acidic fibroblast growth factor protein levels were detected in smooth muscle cells and skeletal myotubes at different time periods after carbaprostacyclin treatment. Furthermore, the selective peroxisome proliferation-activated receptor ß/δ antagonist significantly suppressed carbaprostacyclin-induced acidic fibroblast growth factor protein production. Collectively, our data provide the first morphological and molecular evidence that local delivery of carbaprostacyclin promotes vascular growth in hindlimb ischemia, and that peroxisome proliferation-activated receptor ß/δ signaling plays a critical role in inducing acidic fibroblast growth factor expression.

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.

CD34⁺/M-cadherin⁺ bone marrow progenitor cells promote arteriogenesis in ischemic hindlimbs of ApoE⁻/⁻ mice.

BACKGROUND: Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34⁺/M-cad⁺ BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34⁺/M-cad⁺ BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. METHODS AND FINDINGS: Colony-forming cell assays and flow cytometry analysis showed that CD34⁺/M-cad⁺ BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE⁻/⁻ mice, CD34⁺/M-cad⁺ BMCs alleviated ischemia and significantly improved blood flow compared with CD34⁺/M-cad⁻ BMCs, CD34⁻/M-cad⁺ BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34⁺/M-cad⁺ cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP⁺ CD34⁺/M-cad⁺ cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP⁺ CD34⁺/M-cad⁺ cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34⁺/M-cad⁺ progenitor cells. A cytokine antibody array revealed that CD34⁺/M-cad⁺ cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34⁺/M-cad⁻ cell-conditioned medium. The proangiogenic cytokines secreted by CD34⁺/M-cad⁺ cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34⁺/M-cad⁻ cells during hypoxia. CONCLUSION: CD34⁺/M-cad⁺ BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE⁻/⁻ mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34⁺/M-cad⁺ BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors.

Gene transfer of COX-1 improves lumen size and blood flow in carotid bypass grafts.

BACKGROUND: In autologous saphenous vein grafts, prostacyclin (PGI(1)), a vasoprotective molecule produced by normal endothelial cells, is down-regulated compared with ungrafted saphenous veins and normal carotid arteries. Reduced PGI(2) synthesis may contribute to local platelet deposition, vascular smooth muscle cell (VSMC) accumulation, atherosclerosis, and ultimately failure of venous bypass grafts. We have examined whether gene transfer-mediated overexpression of COX-1 in grafted veins (1) increases PGI(2) and cyclic AMP (cAMP) production, (2) leads to vasodilation and improved local blood flow in the presence of hypercholesterolemia, and (3) reduces neointima formation. MATERIALS AND METHODS: Jugular veins from New Zealand-White rabbits were incubated for 30 min ex vivo with 1 x 10(10) PFU/mL of an adenoviral vector encoding COX-1 (AdCOX-1; n = 10) or empty control (n = 10) and grafted to the carotid arteries. The rabbits were placed on a high-cholesterol diet for 4 w, and blood flow and histomorphometry of the grafts were assessed. RESULTS: In the AdCOX-1 group, blood flow was significantly increased (16.0 +/- 3.3 versus 12.5 +/- 3.3 mL/min; P < 0.05) compared with controls, and luminal area (8.9 +/- 1.4 versus 5.3 +/- 1.2 mm(2); P < 0.01) and outer circumference were larger. In six identically treated rabbits, graft PGI(2) and cAMP synthesis was increased at 72 h in AdCOX-1 compared with controls. CONCLUSION: Our data suggest a 30-min ex vivo exposure of vein grafts to AdCOX-1 increased local synthesis of PGI(2) and cAMP after graft surgery and resulted in better graft lumen and blood flow at 4 w.

Intra-arterial transplantation of adult bone marrow cells restores blood flow and regenerates skeletal muscle in ischemic limbs.

OBJECTIVE: Bone marrow cell therapy promotes angiogenesis, but the cellular fate of bone marrow cells (BMCs) in the absence of immunosuppressant interventions is unclear. We created a model of severe hind limb ischemia to address whether BMCs form new blood vessels or differentiate into other tissues. METHODS AND RESULTS: After ligating the common femoral artery in ApoE knockout mice, we injected either phosphate buffered saline (PBS) or 5 x 10(7) adult unfractionated BMCs obtained from green fluorescent protein-positive mice. Laser Doppler imaging of the ischemic limbs revealed that intra-arterial BMCs significantly increased blood flow recovery in ischemic limbs beginning 21 days after surgery and peaking at 27 days (61.8% +/- 15% vs. 41.9% +/- 13.9%, respectively, for BMCs and PBS, P < .05). The BMCs differentiated into small blood vessels, skeletal myofibers, and supporting membranes, and these changes were associated with increased serum levels of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), transforming growth factor beta (TGFbeta), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNF-alpha). CONCLUSIONS: Adult BMCs injected into ischemic limbs without immunosuppressant therapy differentiated into blood vessels and skeletal myofibers, and this was associated with accelerated blood flow restoration and increased serum levels of VEGF, FGF-2, TGF-beta, IL-4, and TNF-alpha. Skeletal muscle formation may provide benefits beyond angiogenesis to patients with chronic peripheral arterial disease or to patients with low cardiac output states who also suffer from skeletal muscle atrophy.