Effect of perivascular low dose ethanol on rat femoral vessels: Preliminary study.

Vasospasm is one of the important causes of morbidity in free flap and replantation surgery. In secondary Raynaud's phenomenon, nearly half of the patients experience digital ulceration, pain and loss of function at least once in their lifetime. The aim of this study is to investigate the vasodilation effect of ethanol-mediated chemical denervation on peripheral vessels by topical administration. In this study, 27 Wistar albino male rats weighing 250-300 grams were used. The rats were randomly divided into three groups: saline (group S, n = 8), lidocaine (group L, n = 9) and 96% ethanol (group E, n = 9). According to group, 0.1 mL saline, 0.1 mL lidocaine and 0.1 mL ethanol were applied around the rat femoral neurovascular bundle. After the application, on the 0th day and 3th weeks, femoral artery and vein diameters were measured. After 3. weeks, histopathological samples from femoral artery, vein and nerve were evaluated. On the 0th day, the mean diameter of the femoral artery and vein was similar in group E and L and higher than group S. After three weeks, the vasodilatation effect of ethanol was increased in group E. In Group L and S, the vasodilatation effect was lost. Histopathological examination showed that ethanol significantly caused perivascular inflammation and nerve degeneration compared to other agents and did not cause endothelial damage. Vasodilatation obtained by ethanol is a rapid onset and long-lasting effect. It is also inexpensive and effective for peripheral vasodilatation.

Development of an Exercise Training Protocol to Investigate Arteriogenesis in a Murine Model of Peripheral Artery Disease.

Exercise is a treatment option in peripheral artery disease (PAD) patients to improve their clinical trajectory, at least in part induced by collateral growth. The ligation of the femoral artery (FAL) in mice is an established model to induce arteriogenesis. We intended to develop an animal model to stimulate collateral growth in mice through exercise. The training intensity assessment consisted of comparing two different training regimens in C57BL/6 mice, a treadmill implementing forced exercise and a free-to-access voluntary running wheel. The mice in the latter group covered a much greater distance than the former pre- and postoperatively. C57BL/6 mice and hypercholesterolemic ApoE-deficient (ApoE-/-) mice were subjected to FAL and had either access to a running wheel or were kept in motion-restricting cages (control) and hind limb perfusion was measured pre- and postoperatively at various times. Perfusion recovery in C57BL/6 mice was similar between the groups. In contrast, ApoE-/- mice showed significant differences between training and control 7 d postoperatively with a significant increase in pericollateral macrophages while the collateral diameter did not differ between training and control groups 21 d after surgery. ApoE-/- mice with running wheel training is a suitable model to simulate exercise induced collateral growth in PAD. This experimental set-up may provide a model for investigating molecular training effects.

Functional engineered mesenchymal stem cells with fibronectin-gold composite coated catheters for vascular tissue regeneration.

Vascularization of engineered tissues remains one of the key problems. Here, we described a novel approach to promote vascularization of engineered tissues using fibronectin (FN) incorporated gold nanoparticles (AuNP) coated onto catheters with mesenchymal stem cells (MSCs) for tissue engineering. We found that the FN-AuNP composite with 43.5 ppm of AuNP exhibited better biomechanical properties and thermal stability than pure FN. FN-AuNP composites promoted MSC proliferation and increased the biocompatibility. Mechanistically, vascular endothelial growth factor (VEGF) promoted MSC migration on FN-AuNP through the endothelial oxide synthase (eNOS)/metalloproteinase (MMP) signaling pathway. Vascular femoral artery tissues isolated from the implanted FN-AuNP-coated catheters with MSCs expressed substantial CD31 and alpha-smooth muscle actin (α-SMA), displayed higher antithrombotic activity, as well as better endothelialization ability than those coated with all other materials. These data suggested that the implantation of FN-AuNP-coated catheter with MSCs could be a novel strategy for vascular biomaterials applications.

Fluorocopolymer-coated nitinol self-expanding paclitaxel-eluting stent: pharmacokinetics and vascular biology responses in a porcine iliofemoral model.

AIMS: Our aim was to evaluate arterial responses to paclitaxel and a novel fluorocopolymer-coated nitinol low-dose paclitaxel-eluting stent (FP-PES). METHODS AND RESULTS: Human smooth muscle cell (SMC) migration was assessed after exposure to paclitaxel in vitro. For pharmacokinetics and vascular response, FP-PES or bare metal stents (BMS) were implanted in porcine iliofemoral arteries. Paclitaxel significantly inhibited human coronary and femoral artery SMC migration at doses as low as 1 pM. Inhibition was significantly greater for femoral compared with coronary artery SMCs from 1 pM to 1 µM. Pharmacokinetics showed consistent paclitaxel release from FP-PES over the study duration. The peak arterial wall paclitaxel level was 3.7 ng/mg at 10 days, with levels decreasing to 50% of peak at 60 days and 10% at 180 days. Paclitaxel was not detected in blood or remote organs. Arteriogram and histomorphometry analyses showed FP-PES significantly inhibits neointimal proliferation versus BMS at 30 and 90 days. Re-endothelialisation scores were not different between groups. CONCLUSIONS: Paclitaxel affected femoral artery SMC migration at lower concentrations and to a greater degree than it did coronary artery SMCs. The novel FP-PES used in this preclinical study demonstrated a vascular healing response similar to BMS, while significantly inhibiting neointimal formation up to 90 days.

Cathepsin S activity controls ischemia-induced neovascularization in mice.

BACKGROUND: Evidence from human and animal studies has demonstrated elevated levels of the cysteine protease cathepsin S (CatS) in hypoxic atherosclerotic lesions. We hypothesized that silencing of CatS gene would suppress ischemia-induced angiogenic action. METHODS AND RESULTS: Left femoral artery ligation-induced ischemia in mice showed the increased expression and activity of CatS in the ischemic muscle. The CatS-deficiency (CatS(-/-)) mice showed impaired functional recovery following hindlimb ischemia and reduced levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), phospho-Akt (p-Akt), p-endothelial nitric oxide synthase, p-extracellular signal-regulated kinase1/2 (Erk1/2), p-p38 mitogen-activated protein kinase, and vascular endothelial growth factor (VEGF) proteins, as well as reduced levels of matrix metalloproteinase-9 and macrophage infiltration in the ischemic muscles. In vitro, CatS silencing reduced the levels of these targeted essential molecules for angiogenesis and vasculogenesis. Together, the results indicated that the effects of CatS knockdown led to defective endothelial cell invasion, proliferation, and tube formation. This notion was reinforced by the finding that CatS inhibition led to a decreased PPAR-γ level and VEGF/Erk1/2 signaling activation in response to ischemia. CatS(-/-) resulted in decreased circulating EPC-like CD31(+)/c-Kit(+) cells, accompanied by the reduction of the cellular levels of PPAR-γ, p-Akt, and VEGF induced by ischemic stress. Transplantation of bone-marrow-derived mononuclear cells from CatS(+/+) mice restored neovascularization in CatS(-/-) mice. CONCLUSIONS: CatS activity controls ischemia-induced neovascularization partially via the modulation of PPAR-γ and VEGF/Akt signaling activation.

Platform technologies for decellularization, tunic-specific cell seeding, and in vitro conditioning of extended length, small diameter vascular grafts.

The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.

Ex vivo reconstitution of arterial endothelium by embryonic stem cell-derived endothelial progenitor cells in baboons.

There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts downregulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESC-derived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.

The vascular-disrupting agent, combretastatin-A4-phosphate, enhances neurogenic vasoconstriction in rat small arteries.

Combretastatin-A4-phosphate (CA4P/CA4), an anti-cancer drug, induces tumour hypoxia by destabilizing the cytoskeleton in tumour endothelial cells. Hypertensive side effects have been observed. We hypothesized that CA4P/CA4 lead to endothelial dysfunction followed by increased vasoconstriction. Mesenteric small arteries and femoral arteries isolated from male Wistar rats were mounted in microvascular myographs for isometric tension recordings and electrical field stimulation (EFS). Immunoblotting of endothelial nitric oxide synthase (eNOS) was performed on human umbilical vein endothelial cells (HUVECs). CA4P failed per se to change vascular tone. In femoral arteries, endothelial cell removal, l-nitro-arginine (l-NNA, an inhibitor of eNOS) and CA4P enhanced phenylephrine-induced vasoconstriction, while in mesenteric arteries only l-NNA leftward shifted concentration-response curves for phenylephrine. CA4P enhanced vasoconstriction induced by low frequency (0.5-4Hz) EFS in femoral arteries, but not in mesenteric arteries. Neurogenic contractions were inhibited by prazosin, an α(1)-adrenoceptor antagonist. In mesenteric arteries, CA4P and l-NNA inhibited vasorelaxation induced by vanadate, a tyrosine phosphatase inhibitor. CA4P did not affect acetylcholine-induced relaxation. In HUVECs, CA4P increased phosphorylation at eNOS-Thr(495), a negative regulatory site, while the positive phosphorylation site eNOS-Ser(1177) was not affected. CA4 neither influenced the actions of phenylephrine, vanadate nor acetylcholine in femoral and mesenteric arteries. In conclusion, our findings suggest that CA4P, but not CA4, enhances sympathetic adrenergic vasoconstriction probably by increasing eNOS-Thr(495) phosphorylation, in a tissue selective manner. These findings encourage further investigation to show that the hypertension and regional organ ischemia induced by CA4P can be avoided by concomitant treatment with an α(1)-adrenoceptor antagonist.

Recombinant human interleukin-11 treatment enhances collateral vessel growth after femoral artery ligation.

OBJECTIVE: To investigate the role of recombinant human interleukin-11 (rhIL-11) on in vivo mobilization of CD34(+)/vascular endothelial growth factor receptor (VEGFR) 2(+) mononuclear cells and collateral vessel remodeling in a mouse model of hindlimb ischemia. METHODS AND RESULTS: We observed that treatment of Sv129 mice with continuous infusion of 200-µg/kg rhIL-11 per day led to in vivo mobilization of CD34(+)/VEGFR2(+) cells that peaked at 72 hours. Sv129 mice pretreated with rhIL-11 for 72 hours before femoral artery ligation showed a 3-fold increase in plantar vessel perfusion, leading to faster blood flow recovery; and a 20-fold increase in circulating CD34(+)/VEGFR2(+) cells after 8 days of rhIL-11 treatment. Histologically, experimental mice had a 3-fold increase in collateral vessel luminal diameter after 21 days of rhIL-11 treatment and a 4.4-fold influx of perivascular CD34(+)/VEGFR2(+) cells after 8 days of therapy. Functionally, rhIL-11-treated mice showed better hindlimb appearance and use scores when compared with syngeneic mice treated with PBS under the same experimental conditions. CONCLUSIONS: These novel findings show that rhIL-11 promotes in vivo mobilization of CD34(+)/VEGFR2(+) mononuclear cells, enhances collateral vessel growth, and increases recovery of perfusion after femoral artery ligation. Thus, rhIL-11 has a promising role for development as an adjunctive treatment of patients with peripheral vascular disease.

Adipose tissue-derived stem cells inhibit neointimal formation in a paracrine fashion in rat femoral artery.

Subcutaneous adipose tissue contains a lot of stem cells [adipose-derived stem cells (ASCs)] that can differentiate into a variety of cell lineages. In this study, we isolated ASCs from Wistar rats and examined whether ASCs would efficiently differentiate into vascular endothelial cells (ECs) in vitro. We also administered ASCs in a wire injury model of rat femoral artery and examined their effects. ASCs expressed CD29 and CD90, but not CD34, suggesting that ASCs resemble bone marrow-derived mesenchymal stem cells. When induced to differentiate into ECs with endothelial growth medium (EGM), ASCs expressed Flt-1, but not Flk-1 or mature EC markers such as CD31 and vascular endothelial cadherin. ASCs produced angiopoietin-1 when they were cultured in EGM. ASCs stimulated the migration of EC, as assessed by chemotaxis assay. When ASCs that were cultured in EGM were injected in the femoral artery, the ASCs potently and significantly inhibited neointimal formation without being integrated in the endothelial layer. EGM-treated ASCs significantly suppressed neointimal formation even when they were administered from the adventitial side. ASC administration significantly promoted endothelial repair. These results suggested that although ASCs appear to have little capacity to differentiate into mature ECs, ASCs have the potential to secrete paracrine factors that stimulate endothelial repair. Our results also suggested that ASCs inhibited neointimal formation via their paracrine effect of stimulation of EC migration in situ rather than the direct integration into the endothelial layer.

KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium.

The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH(2)-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-alpha-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

Cyclic stretch controls the expression of CD40 in endothelial cells by changing their transforming growth factor-beta1 response.

BACKGROUND: CD40 is a costimulatory molecule that acts as a central mediator of various immune responses, including those involved in the progression of atherosclerosis. Correspondent to its function, CD40 is present not only on many immune cells, such as antigen-presenting cells and T cells, but also on nonimmune cells, such as endothelial cells. METHODS AND RESULTS: Ex vivo analyses in mice revealed that CD40 is strongly expressed in distinct venous and capillary but not arterial endothelial cell populations. Therefore, we analyzed to what extent determinants of an arterial environment control CD40 expression in these cells. In vitro studies indicated that the presence of smooth muscle cells or exposure to cyclic stretch significantly downregulates CD40 expression in human endothelial cells. Interestingly, endothelial cells cocultured with smooth muscle cells upregulated CD40 expression in response to cyclic stretch through a transforming growth factor-beta1/activin-receptor-like kinase-1 (Alk-1)-dependent mechanism. To corroborate that this mechanism also operates in arteries in vivo, we analyzed the expression of Alk-1 and CD40 at atherosclerosis-prone sites of the mouse aorta that also appear to be exposed to increased stretch. In wild-type mice, both Alk-1 and CD40 revealed a comparably heterogeneous expression pattern along the aortic arch that matched those sites in low-density lipoprotein-receptor-deficient mice where atherosclerotic lesions develop. CONCLUSIONS: Cyclic stretch thus increases the abundance of CD40 in endothelial cells through transforming growth factor-beta1/Alk-1 signaling. This mechanism in turn may be responsible for the heterogeneous expression of CD40 at arterial bifurcations or curvatures and would support a site-specific proinflammatory response that is typical for the early phase of atherosclerosis.

Stem cell factor attenuates vascular smooth muscle apoptosis and increases intimal hyperplasia after vascular injury.

OBJECTIVE: Stem cell factor (SCF) through its cognate receptor, the tyrosine kinase c-kit, promotes survival and biological functions of hematopoietic stem cells and progenitors. However, whether SCF/c-kit interactions exacerbate intimal hyperplasia through attenuating VSMC apoptosis induced by vascular injury has not been thoroughly investigated. METHODS AND RESULTS: VSMCs were stimulated with serum deprivation and H2O2 to induce apoptosis. The transcription of c-kit mRNA and the expression of the c-kit protein by VSMCs were estimated by Q-polymerase chain reaction and Western blotting, respectively. The interactions of SCF and c-kit were investigated by in vitro and in vivo experiments. In vitro, H2O2 stimulation significantly induced apoptosis of VSMCs as evidenced by the 3- and 3.2-fold increases of cleaved caspase-3 compared with those in the control group by Western blot and flow cytometric analyses, respectively (P<0.01). Stimulation of apoptosis also caused 3.5- and 9-fold increases in c-kit mRNA transcription and protein expression, respectively, by VSMCs compared with those in the control group. Administration of SCF (10 to 1000 ng/mL) significantly lowered the amount of cleaved caspase-3 in H2O2-treated VSMCs (P<0.01). Specifically, SCF exerted this effect through activating Akt, followed by increasing Bcl-2 and then inhibiting the release of cytochrome-c from the mitochondria to the cytosol. In vivo, the mouse femoral artery was injured with a wire in SCF mutant (Sl/Sl(d)), c-kit mutant (W/W(v)), and colony control mice. In colony control mice, confocal microscopy demonstrated that the wire-injury generated a remarkable activation of caspase-3 on medial VSMCs, coinciding with upregulation of c-kit expression. The wire-injury also caused an increase in the expression of SCF on surviving medial VSMCs and cells in the adventitia. The upregulated c-kit expression in the vessel wall also facilitated homing by circulating SCF+ cells. Compared with colony control mice, vascular injury in SCF mutant and c-kit mutant mice caused a higher number of apoptotic VSMCs on day 14 and a lower number of proliferating cells, and resulted in significantly less neointimal formation (P<0.01) on day 28. CONCLUSIONS: The interactions between SCF and the c-kit receptor play an important role in protecting VSMCs against apoptosis and in maintaining intimal hyperplasia after vascular injury.

Thrombotic effect of purposeful back-wall stitch for different suture locations and vessels in rats.

Many microsurgeons experience inadvertent back-wall stitch as a cause of immediate anastomotic failure. To investigate this factor as a possible cause of delayed arterial anastomotic failure that does not appear in the operation room, a purposeful back-wall stitch (PBWS) model of microarterial anastomosis was applied in various configurations on femoral and carotid arteries of rats. Carotid (n = 28) and femoral (n = 28) artery groups were equally divided into 4 different subgroups according to the type of PBWS. Control subgroups had no PBWS. Thirty-degree, 60-degree, and 90-degree subgroups had PBWS located at 30, 60, and 90 degrees, respectively. Patencies were assessed at 60 minutes and 24 hours. Immediate milking test demonstrated normal anterograde refilling in all anastomoses. PBWS increased thrombosis in femoral arteries, while it did not increase it in the carotid at either evaluation times. The only significant difference was between 90 degrees PBWS and the control subgroup irrespective of the vessel factor in 24 hours. Histologic examination supported absence of thrombosis, as suggested by surgical examination. This may show that small-sized arteries are more inclined to thrombosis compared with larger ones and the thrombosis risk increases as the inadvertent back-wall stitch is more centrally located. Contrary to general notion that the inadvertent back-wall stitch causes immediate thrombosis, thromboses later than 1 hour, and even patent anastomoses in 24 hours, were observed in femoral and carotid artery groups. This study suggests that the inadvertent back-wall stitch should also be considered as a possible cause of late anastomotic problems.

Targeted delivery of bone marrow mononuclear cells by ultrasound destruction of microbubbles induces both angiogenesis and arteriogenesis response.

OBJECTIVE: Ultrasound (US)-mediated destruction of contrast microbubbles causes capillary rupturing that stimulates arteriogenesis, whereas intramuscular implantation (im) of bone marrow mononuclear cells (BM-MNCs) induces angiogenesis. We therefore studied whether US-targeted microbubble destruction combined with transplantation of BM-MNCs can enhance blood flow restoration by stimulating both angiogenesis and arteriogenesis. METHODS AND RESULTS: US-mediated destruction of phospholipid-coated microbubbles was applied onto ischemic hindlimb muscle and subsequently BM-MNCs were transfused. A significant enhancement in blood flow recovery after Bubble+US+BM-MNC infusion (34% increase, P<0.05) was observed compared with Bubble+US (25%). The ratio of capillary/muscle fiber increased by Bubble+US+BM-MNC-i.v (260%, P<0.01) than that in the Bubble+US group (172%), into which BM-MNCs were incorporated (angiogenesis). Smooth muscle alpha-actin-positive arterioles were also increased, and angiography showed augmented collateral vessel formation (arteriogenesis). Platelet-derived proinflammatory factors activated by Bubble+US induces the expression of adhesion molecules (P-selectin and ICAM-1), leading to the attachment of transplanted BM-MNCs on the endothelium. Flow assay confirmed that the platelet-derived factors cause the adhesion of BM-MNCs onto endothelium under laminar flow. CONCLUSIONS: This study demonstrates that the targeted delivery of BM-MNCs by US destruction of microbubbles enhances regional angiogenesis and arteriogenesis response, in which the release of platelet-derived proinflammatory factors activated by Bubble+US play a key role in the attachment of transplanted BM-MNCs onto the endothelial layer.

Potent inhibitory effect of sirolimus on circulating vascular progenitor cells.

BACKGROUND: Neointimal hyperplasia is the major cause of in-stent restenosis (ISR). The sirolimus-eluting stent (SES) has emerged as a promising therapy to prevent ISR; however, the exact mechanism by which locally delivered sirolimus, an immunosuppressive agent, prevents ISR remains unknown. Recent evidence suggests that circulating progenitor cells may contribute to neointimal formation. METHODS AND RESULTS: Mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers. Smooth muscle (SM)-like cells outgrew from the culture of MNCs (1x10(6)) in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas endothelial cell-like cells were obtained in the presence of vascular endothelial growth factor. Sirolimus potently inhibited SM-like cell outgrowth. The number of SM-like cells was significantly reduced at a concentration as low as 0.1 ng/mL (15.9+/-5.8% of control, P<0.001). Sirolimus also exerted an inhibitory effect on endothelial cell-like cells that originated from MNCs. Wire-mediated vascular injury was induced in femoral arteries of bone marrow chimeric mice. Either vehicle or sirolimus was administered locally to the perivascular area of the injured arteries. Sirolimus significantly reduced neointima hyperplasia at 4 weeks (intima/media ratio 2.0+/-0.3 versus 1.0+/-0.2, P<0.05) with a decreased number of bone marrow-derived SM-like cells and hematopoietic cells in the lesion. Reendothelialization was retarded in the arteries treated with sirolimus. CONCLUSIONS: The potent inhibitory effects of sirolimus on circulating smooth muscle progenitor cells may mediate the clinical efficacy of SES, at least in part. Sirolimus potentially may affect reendothelialization after stent implantation.

Expression of vascular endothelial growth factor receptor-2 or Tie-2 on peripheral blood cells defines functionally competent cell populations capable of reendothelialization.

BACKGROUND: Receptor tyrosine kinases that include vascular endothelial growth factor (VEGFR)-1, VEGFR-2, and Tie-2 regulate cardiovascular development and physiological and pathological angiogenesis. We were interested in the phenotypic and functional characterization of peripheral blood cells expressing these receptors and their therapeutic potential in vascular injury. METHODS AND RESULTS: VEGFR-1+, VEGFR-2+, and Tie-2+ cells constituted approximately 3.0+/-0.2%, 0.8+/-0.5%, and 2.0+/-0.3%, respectively, of the total population of mononuclear cells in blood. Phenotypic analysis demonstrated that all 3 cell populations mainly expressed markers of monocytic/macrophage lineage. Only VEGFR-2+ and Tie-2+ cells phenotypically, morphologically, and functionally differentiated to endothelial cells after culture, whereas VEGFR-1+ cells did not. None of the cell types proliferated in vitro. Only freshly isolated VEGFR-2+ or Tie-2+ cells but not VEGFR-2- or Tie-2- cell populations significantly contributed to efficient endothelialization of balloon-injured femoral arteries of nude mice. Furthermore, these cells also differentiated into -actin-positive smooth muscle cells. Administration of bromodeoxyuridine to animals transplanted with human endothelial progenitor cells showed that VEGFR-2+ and Tie-2+ cells proliferated in vivo. CONCLUSIONS: These data demonstrate that expression of VEGFR-2 and/or Tie-2 on peripheral blood cells defines functionally competent cell populations that proliferate in vivo and that contribute to reendothelialization. These findings may have implications for a cell-based approach in vascular diseases.

Healing of implanted expanded polytetrafluoroethylene vascular access grafts with different internodal distances: a histologic study in dogs.

OBJECTIVE: We assessed characteristics of healing, over time, of two types of expanded polytetrafluoroethylene grafts. STUDY DESIGN: An experimental histological study in dogs. METHODS: The graft types studied had the same internal diameter (5 mm) but different internodal distances. In one, the internodal distance was 60 microm in the external surface and 20 microm in the luminal surface. In the other, the internodal distance was 30 microm throughout the material. Sixteen grafts of each type were implanted between the femoral artery and vein in 16 dogs; explanted 1, 2, 4 or 12 weeks later; and examined histologically. RESULTS: In both graft types, infiltrating-cell density and maximum cell-penetration depth increased significantly between 1 and 2 weeks after implantation, but no significant increases occurred after 2 weeks. The number of inflammatory cells peaked 1 week after implantation and decreased significantly by 2 weeks. Subsequently, there were no significant changes in inflammatory cell numbers, suggesting that the inflammatory phase was over by 2 weeks after implantation and the grafts had become attached to surrounding tissue. There were no significant differences between the two graft types in cell density, cell-penetration depth, or number of inflammatory cells at any assessment time. CONCLUSION: Our results provide histologic support for guidelines recommending that synthetic vascular grafts for hemodialysis access should not be cannulated until 2 weeks after implantation. Since increasing the internodal distance to 60 microm in the external surface had no effect on graft healing, methods other than manipulation of internodal distance should be used in developing a graft suitable for early cannulation.

Cellular remodeling of depopulated bovine ureter used as an arteriovenous graft in the canine model.

BACKGROUND: One of the greatest challenges in hemodialysis access surgery is improving the durability of prosthetic grafts caused by structural deterioration. The depopulated bovine ureter SynerGraft (SG) (CryoLife, Inc) is a tissue-engineered vascular graft processed to remove the xenograft cells while maintaining an unfixed connective tissue matrix capable of autologous cell repopulation by the recipient. STUDY DESIGN: Nineteen 6-mm diameter bovine ureter SG conduits were implanted in 12 dogs as arteriovenous grafts between the carotid artery and jugular vein (n = 11) or between the femoral artery and vein (n = 8). Performance of these biologic conduits was compared with that of 15 IMPRA (Bard) ePTFE grafts implanted in 9 dogs, including 9 arteriovenous grafts between the carotid artery and jugular vein and 6 femoral artery to femoral vein grafts. After 14 days, the grafts were accessed once weekly. Histologic and immunohistochemical analyses were performed on grafts explanted between 10 to 60 weeks. RESULTS: The 6- and 12-month primary patency rates of the bovine SG were 72.6% and 58.6%, respectively, compared with 6- and 12-month primary patency for ePTFE conduits of 57.4% and 57.4%, respectively. None of the bovine SG grafts became infected, but synthetic conduits became infected within 54 days of implantation. At 10 weeks, bovine ureter SG conduit showed fibroblast cell migration and proliferation with incorporation into the surrounding subcutaneous tissue, and elongated cells expressing the contractile protein smooth muscle actin were also observed. After 24 weeks, procollagen synthesis was demonstrated in the fully colonized graft matrix. The ePTFE grafts had no evidence of cellular ingrowth and an absence of endothelium. CONCLUSIONS: The bovine SG was appropriately remodeled to its host environment through an organized process of recellularization and neovascularization. The absence of infection, similar patency rates, and cell repopulation of the matrix warrant further investigation.

Role of bone marrow-derived progenitor cells in cuff-induced vascular injury in mice.

OBJECTIVE: Arterial injury results in vascular remodeling associated with proliferation and migration of smooth muscle cells (SMCs) and the development of intimal hyperplasia, which is a critical component of restenosis after angioplasty of human coronary arteries and an important feature of atherosclerotic lesions. However, the origin of SMCs and other cells in the development of vascular remodeling is not yet fully understood. METHODS AND RESULTS: We utilized a cuff-induced vascular injury model after transplantation of the bone marrow (BM) from green fluorescent protein (GFP)-transgenic mice. We found that macrophages were major cells recruited to the adventitia of the vascular injury lesion along with SMCs and endothelial cells (ECs). While investigating whether those cells are derived from the donor, we found that most of the macrophages were GFP-positive, and some of the SMCs and ECs were also GFP-positive. Administration of the anti-c-fms antibody resulted in a marked decrease in macrophages and a relative increase of SMCs, while administration of antibodies against the platelet-derived growth factor receptor-beta caused a prominent decrease in SMCs and a relative increase in macrophages. CONCLUSIONS: The current study indicates that BM-derived cells play an important role in vascular injury, and that differentiation of macrophages and SMCs might be dependent on each other.