Genetic lineage tracing analysis of c-kit+ stem/progenitor cells revealed a contribution to vascular injury-induced neointimal lesions.

AIMS: Accumulating evidence indicates the presence of vascular stem/progenitor cells that may play a role in endothelial repair and lesion formation in the injured artery, in which c-kit+ stem/progenitor cells have been reported to differentiate into endothelial and smooth muscle cells in vitro and in ischemic tissue. In this study, we investigated whether and how endogenous c-kit+ stem/progenitor cells contribute to vascular injury and neointima formation in vivo. METHODS AND RESULTS: We created Kit-CreERxRosa26-RFP mice and performed genetic lineage tracing analysis of c-kit+ stem/progenitor cells in injury-induced neointima formation in vivo. We provide direct evidence that endogenous c-kit+ stem/progenitor cells minimally differentiate into endothelial or smooth muscle cells facilitating vascular repair, but predominantly generate monocytes/macrophages and granulocytes contributing to vascular immuno-inflammatory response to endothelial injury. Although c-kit+ cells reside in both bone marrow and vessel wall, bone marrow transplantation data indicate that bone marrow-derived c-kit+ cells are the main source for enhancing neointima formation. Furthermore, treatment of ACK2, a c-kit receptor antagonizer, attenuates neointimal hyperplasia after injury at least in part by depleting c-kit+ cells and their generated progeny. CONCLUSIONS: c-kit+ stem/progenitor cells are not a main source for endothelial regeneration and smooth muscle accumulation of the large artery injury, but a plausible interventional approach to reduce vascular immuno-inflammatory response and subsequently to ameliorate vascular lesions.

Vasa Vasorum Angiogenesis: Key Player in the Initiation and Progression of Atherosclerosis and Potential Target for the Treatment of Cardiovascular Disease.

Plaque microvascularization and increased endothelial permeability are key players in the development of atherosclerosis, from the initial stages of plaque formation to the occurrence of acute cardiovascular events. First, endothelial dysfunction and increased permeability facilitate the entry of diverse inflammation-triggering molecules and particles such as low-density lipoproteins into the artery wall from the arterial lumen and vasa vasorum (VV). Recognition of entering particles by resident phagocytes in the vessel wall triggers a maladaptive inflammatory response that initiates the process of local plaque formation. The recruitment and accumulation of inflammatory cells and the subsequent release of several cytokines, especially from resident macrophages, stimulate the expansion of existing VV and the formation of new highly permeable microvessels. This, in turn, exacerbates the deposition of pro-inflammatory particles and results in the recruitment of even more inflammatory cells. The progressive accumulation of leukocytes in the intima, which trigger proliferation of smooth muscle cells in the media, results in vessel wall thickening and hypoxia, which further stimulates neoangiogenesis of VV. Ultimately, this highly inflammatory environment damages the fragile plaque microvasculature leading to intraplaque hemorrhage, plaque instability, and eventually, acute cardiovascular events. This review will focus on the pivotal roles of endothelial permeability, neoangiogenesis, and plaque microvascularization by VV during plaque initiation, progression, and rupture. Special emphasis will be given to the underlying molecular mechanisms and potential therapeutic strategies to selectively target these processes.

Self-assembled Collagen-Fibrin Hydrogel Reinforces Tissue Engineered Adventitia Vessels Seeded with Human Fibroblasts.

Efforts for tissue engineering vascular grafts focuses on the tunica media and intima, although the tunica adventitia serves as the primary structural support for blood vessels. In surgery, during endarterectomies, surgeons can strip the vessel, leaving the adventitia as the main strength layer to close the vessel. Here, we adapted our recently developed technique of forming vascular tissue rings then stacking the rings into a tubular structure, to accommodate human fibroblasts to create adventitia vessels in 8 days. Collagen production and fibril cross-linking was augmented with TGF-ß and ascorbic acid, significantly increasing tensile strength to 57.8 ± 3.07 kPa (p = 0.008). Collagen type I gel was added to the base fibrin hydrogel to further increase strength. Groups were: Fibrin only; 0.7 mg/ml COL; 1.7 mg/ml COL; and 2.2 mg/ml COL. The 0.7 mg/ml collagen rings resulted in the highest tensile strength at 77.0 ± 18.1 kPa (p = 0.015). Culture periods of 1-2 weeks resulted in an increase in extracellular matrix deposition and significantly higher failure strength but not ultimate tensile strength. Histological analysis showed the 0.7 mg/ml COL group had significantly more, mature collagen. Thus, a hydrogel of 0.7 mg/ml collagen in fibrin was ideal for creating and strengthening engineered adventitia vessels.

Attenuation of ductus arteriosus intimal thickening in preterm sheep twins compared with singletons.

Preterm twins have a higher morbidity rate of patent ductus arteriosus (PDA) than do singletons. However, the effect of multiple births on maturation of the ductus arteriosus (DA) has not been reported. Because intimal thickening (IT) is required for DA anatomical closure, we examined IT development in the DA of preterm twins and singletons. Sheep DA tissues obtained from preterm fetuses were subjected to elastica van Gieson staining to evaluate IT. The total IT score in each DA was the sum of the IT scores obtained from six evenly divided parts of the DA, which was positively correlated with gestational ages in singletons. Total IT scores were smaller in preterm twins than in singletons, although no difference in gestational age, birth weight, or gender ratio was observed. These data suggest that IT development of the DA is attenuated in sheep preterm twins, which may affect the higher morbidity of PDA.

Tropoelastin inhibits intimal hyperplasia of mouse bioresorbable arterial vascular grafts.

Neointimal hyperplasia, which results from the activation, proliferation and migration of vascular smooth muscle cells (SMCs), is a detrimental condition for vascular stents or vascular grafts that leads to stenosis. Preventing neointimal hyperplasia of vascular grafts is critically important for the success of arterial vascular grafts. We hypothesized that tropoelastin seeding onto the luminal surface of the graft would prevent neointimal hyperplasia through suppressing neointimal smooth muscle cell proliferation. In this study, we investigated the efficacy of tropoelastin seeding in preventing neointimal hyperplasia of bioresorbable arterial vascular grafts. Poly (glycolic acid) (PGA) fiber mesh coated with poly (l-lactic-co-ε-caprolactone) (PLCL) scaffolds reinforced by poly (l-lactic acid) (PLA) nano-fibers were prepared as bioresorbable arterial grafts. Tropoelastin was then seeded onto the luminal surface of the grafts. Tropoelastin significantly reduced the thickness of the intimal layer. This effect was mainly due to a substantial reduction the number of cells that stained positive for SMC (α-SMA) and PCNA in the vessel walls. Mature elastin and collagen type I and III were unchanged with tropoelastin treatment. This study demonstrates that tropoelastin seeding is beneficial in preventing SMC proliferation and neointimal hyperplasia in bioresorbable arterial vascular grafts. STATEMENT OF SIGNIFICANCE: Small resorbable vascular grafts can block due to the over-proliferation of smooth muscle cells in neointimal hyperplasia. We show here that the proliferation of these cells is restricted in this type of graft. This is achieved with a simple dip, non-covalent coating of tropoelastin. It is in principle amendable to other grafts and is therefore an attractive process. This study is particularly significant because: (1) it shows that smooth muscle cell proliferation can be reduced while still accommodating the growth of endothelial cells, (2) small vascular grafts with an internal diameter of less than 1mm are amenable to this process, and (3) this process works for resorbable grafts.

PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.

Carotid intima-media thickness at age 30, birth weight, accelerated growth during infancy and breastfeeding: a birth cohort study in Southern Brazil.

OBJECTIVE: To examine the relationship between carotid intima-media thickness (IMT) at age 30 and birth characteristics, growth during infancy, and breastfeeding duration, among subjects who have been prospectively followed since birth. METHODS AND RESULTS: In 1982, all births in the city of Pelotas, southern Brazil, were identified and those children (n = 5,914) whose families lived in the urban area of the city have been followed and evaluated at several time points. The cohort participants were evaluated in 2012-13, and IMT was measured at the posterior wall of the right and left common carotid arteries in longitudinal planes using ultrasound imaging. We obtained valid IMT measurements for 3,188 individuals. Weight-for-age z-score (WAZ) at age 2 years, weight-for-height z-score (WHZ) at age 4, height-for-age z-score (HAZ) at 4 years, WAZ at age 4 and relative conditional weight at 4 years were positively associated with IMT, even after controlling for confounding variables. The beta-coefficient associated with ≥ 1 s.d. WAZ at age 2 (compared to those with a <-1 s.d.) was 3.62 µm (95% CI 0.86 to 6.38). The beta-coefficient associated with ≥ 1 s.d. WHZ at 4 (in relation to <-1 s.d) was 3.83 µm (95% CI 0.24 to 7.42). For HAZ at 4, the beta-coefficient for ≥ 1 s.d. in relation to <-1 s.d. was 4.19 µm (95% CI 1.14 to 7.25). For WAZ at 4, the beta-coefficient associated with ≥ 1 s.d. in relation to <-1 s.d. was 4.28 µm (95% CI 1.59 to 6.97). The beta-coefficient associated with conditional weight gain at age 2-4 was 1.26 µm (95% CI 0.49 to 2.02). CONCLUSION: IMT at age 30 was positively associated with WAZ at age 2 years, WHZ at age 4, HAZ at age 4, WAZ at age 4 and conditional weight gain at age 4 years.

Lentiviral vector mediated expression of Bax and hepatocyte growth factor inhibits vein graft thickening in a rabbit vein graft model.

Vein graft failure caused by vein graft thickening of the arterialized vein after bypass surgery is a main problem in clinical vascular surgery. Gene therapy is increasingly being recognized as a relevant treatment option for vein graft failure. In this study, we aimed to develop a novel recombinant lentivirus for the delivery of hepatocyte growth factor (HGF) and Bax in a rabbit vein graft model of bypass grafting. A bypass model was made in rabbits using the right jugular vein interposed end-to-end to the ipsilateral carotid artery. A lentivirus vector harboring HGF and Bax cDNAs (Lenti-HGF-Bax) was constructed and transduced into the venous grafts. Vein grafts were stained with hematoxilyn and eosin, and Masson. HGF and Bax expression in vein grafts was detected by immunohistochemical and Western blot analysis. Our results showed that vein graft thickening was reduced by 47.2 ± 7.4% in lenti-HGF-Bax treated rabbits, compared to controls. Meanwhile, the ratio of intima/media area was reduced in lentil-HGF-Bax treated rabbits, compared to controls. The number of HGF and Bax positive cells was increased in vein grafts from rabbits treated by lenti-HGF-Bax, compared to those from controls. Furthermore, protein levels of HGF and Bax were both significantly increased in grafts derived from rabbits treated by lenti-HGF-Bax, compared to those from control. In conclusion, Lenti-HGF-Bax inhibits vein graft thickening in vein grafts and is a promising agent for preventing vein graft failure.

Morphometric study of aortic wall parameters evolution in newborn and child.

The largest artery in the human body, intimately connected to the heart, aorta is usually regarded as the major source of oxygenated blood for the circulatory system. The three concentric layers, which surround the aortic lumen-the tunics intima, media and adventitia, transform the aorta in a large elastic duct, which is irregular calibrated according to its segments. The special aortic distensibility is facilitated by its elastic circumferential lamellar complex. Any disturbance of its structural components is able to interfere with its normal and vital activity. Our study intends to reveal that the development of elastic lamellae should be regarded not only as an indispensable step for the aortic wall configuration, but also like a process in a firm connection with the rest of aortic wall components. The transition from intrauterine life to a new stage of life, childhood, has to determine an adequate adaptation of almost all the components of aortic wall, in order to sustain a consistent pulsatile blood flow. Stereological quantitative analysis of thoracic aortic fragments prelevated from newborns and children was performed in order to estimate the dynamic of vascular wall increase. We first estimated the general configuration of the thoracic aortic wall, quantifying the principal constituents; the connective tissue profile, investigated through its main elements, collagen and elastic fibers, supports the idea that each type of fiber has a distinct evolution in different groups of ages and has to be correlated with their involvement in maintaining of the aortic wall mechanical properties. Elastic fibers percentage volume was increased in both examined groups, with a small difference reported in children aorta, while collagen fibers exhibit a slow increase in children aorta. Our morphometric quantitative assessment suggests that further studies have to draw of in a precisely manner the outline of the secretory well defined function of vascular smooth muscle cells; the elucidation of the manner in which the secretory pathway for each type of fiber becomes fully adapted to every stage of aortic development will allow a new perspective in aortic pathology.

Adipose-derived factor CTRP9 attenuates vascular smooth muscle cell proliferation and neointimal formation.

Obesity is closely associated with the progression of vascular disorders, including atherosclerosis and postangioplasty restenosis. C1q/TNF-related protein (CTRP) 9 is an adipocytokine that is down-regulated in obese mice. Here we investigated whether CTRP9 modulates neointimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation in vivo and in vitro. Left femoral arteries of wild-type (WT) mice were injured by a steel wire. An adenoviral vector expressing CTRP9 (Ad-CTRP9) or ß-galactosidase as a control was intravenously injected into WT mice 3 d before vascular injury. Delivery of Ad-CTRP9 significantly attenuated the neointimal thickening and the number of bromodeoxyuridine-positive proliferating cells in the injured arteries compared with that of control. Treatment of VSMCs with CTRP9 protein attenuated the proliferative and chemotactic activities induced by growth factors including platelet-derived growth factor (PDGF)-BB, and suppressed PDGF-BB-stimulated phosphorylation of ERK. CTRP9 treatment dose-dependently increased cAMP levels in VSMCs. Blockade of cAMP-PKA pathway reversed the inhibitory effect of CTRP9 on DNA synthesis and ERK phosphorylation in response to PDGF-BB. The present data indicate that CTRP9 functions to attenuate neointimal formation following vascular injury through its ability to inhibit VSMC growth via cAMP-dependent mechanism, suggesting that the therapeutic approaches to enhance CTRP9 production could be valuable for prevention of vascular restenosis after angioplasty.

RAGE mediates accelerated diabetic vein graft atherosclerosis induced by combined mechanical stress and AGEs via synergistic ERK activation.

AIMS/HYPOTHESIS: Diabetes with hypertension rapidly accelerates vascular disease, but the underlying mechanism remains unclear. We evaluated the hypothesis that the receptor of advanced glycation end products (RAGE) might mediate combined signals initiated by diabetes-related AGEs and hypertension-induced mechanical stress as a common molecular sensor. METHODS: In vivo surgical vein grafts created by grafting vena cava segments from C57BL/6J mice into the common carotid arteries of streptozotocin (STZ)-treated and untreated isogenic mice for 4 and 8 weeks were analyzed using morphometric and immunohistochemical techniques. In vitro quiescent mouse vascular smooth muscle cells (VSMCs) with either knockdown or overexpression of RAGE were subjected to cyclic stretching with or without AGEs. Extracellular signal-regulated kinase (ERK) phosphorylation and Ki-67 expression were investigated. RESULTS: Significant increases in neointimal formation, AGE deposition, Ki-67 expression, and RAGE were observed in the vein grafts of STZ-induced diabetic mice. The highest levels of ERK phosphorylation and Ki-67 expression in VSMCs were induced by simultaneous stretch stress and AGE exposure. The synergistic activation of ERKs and Ki-67 in VSMCs was significantly inhibited by siRNA-RAGE treatment and enhanced by over-expression of RAGE. CONCLUSION: RAGE may mediate synergistically increased ERK activation and VSMC proliferation induced by mechanical stretching with and without AGEs. It may serve as a common molecular bridge between the two, accelerating vascular remodeling. This study provides potential drug targets and novel therapeutic strategies for the treatment of vascular diseases resulting from diabetes with hypertension.

Apigenin attenuates neointima formation via suppression of vascular smooth muscle cell phenotypic transformation.

Abnormal proliferation, migration, and phenotypic modulation of vascular smooth muscle cells (VSMCs) are critical factors in neointima formation during restenosis. The purpose of this study is to determine the efficacy and possible cell signaling mechanisms of apigenin in VSMC activation induced by platelet-derived growth factor (PDGF)-BB and injury-induced neointima formation. Our data revealed a dose-dependent apigenin inhibition of PDGF-BB-induced proliferation of VSMCs by arresting cells in G0/G1-phase of the cell cycle as determined using 5-bromo-2'-deoxyuridine incorporation and flow cytometry. This was associated with the inhibition of cyclin-dependent kinase (CDK) 4,6 expression and an increase in p27Kip1 levels in PDGF-stimulated VSMCs. Moreover, apigenin was also found to regulate PDGF-induced migration and expression of smooth-muscle-specific contractile markers. Mechanistically, the PDGF-BB-induced phosphorylation of PDGF-receptor ß (PDGF-Rß), Akt/glycogen synthase kinase(GSK)3ß, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3) is negatively modulated by apigenin. For the in vivo studies using a mouse carotid arterial injury model, the administration of apigenin resulted in a significant inhibition of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells. These results demonstrate that apigenin can suppress PDGF-induced VSMC activation and neointima hyperplasia after vascular injury; these beneficial effects are probably the result of the blockade of PDGF-Rß phosphorylation and its downstream signal transduction, including the Akt/GSK-3ß, ERK1/2, and STAT3 pathways. The results suggest that apigenin may be a potential therapeutic candidate for the prevention of restenosis.

Polymer-free biolimus a9-coated stent demonstrates more sustained intimal inhibition, improved healing, and reduced inflammation compared with a polymer-coated sirolimus-eluting cypher stent in a porcine model.

BACKGROUND: Drug-eluting stents effectively reduce restenosis but may increase late thrombosis and delayed restenosis. Persistent polymer, the drug, or a combination of both could be responsible. Local delivery of Biolimus A9, a rapamycin derivative, from a polymer-free BioFreedom stent (Biosensors International) may prevent these complications. METHODS AND RESULTS: We compared high-dose (HD) (225 microg/14 mm Biolimus A9) and low-dose (LD) (112 microg/14 mm Biolimus A9) BioFreedom stents with a polymer-coated sirolimus-eluting Cypher stent (SES) and a bare-metal stent (BMS) at 28 days and 180 days in an overstretch coronary mini-swine model with histomorphometric and histological analysis. At 28 days, there was a reduction in neointimal proliferation by HD, LD, and SES compared with BMS (neointimal thickness: HD, 0.080+/-0.032; LD, 0.085+/-0.038; SES, 0.064+/-0.037; BMS, 0.19+/-0.111 mm; P<0.001; BMS > HD/LD/SES). At 180 days, both BioFreedom stents were associated with reduced neointimal proliferation, whereas SES exhibited increased neointima (neointimal thickness: HD, 0.12+/-0.034; LD, 0.10+/-0.040; SES, 0.20+/-0.111; BMS, 0.17+/-0.099 mm; P<0.001; SES > HD/LD; BMS > LD). At 180 days, BioFreedom stents showed decreased fibrin and inflammation, including granuloma and giant cells, compared with SES. CONCLUSIONS: The polymer-free Biolimus A9-coated stent demonstrates equivalent early and superior late reduction of intimal proliferation compared with SES in a porcine model. After implantation of BioFreedom stent, delayed arterial healing was minimal, and there was no increased inflammation at 180 days compared with SES implantation. The use of polymer-free stents may have a potential long-term benefit over traditional polymeric-coated drug-eluting stents.

Periadventitial rapamycin-eluting microbeads promote vein graft disease in long-term pig vein-into-artery interposition grafts.

BACKGROUND: Neointima formation and atherosclerosis compromise long-term graft patency in aortocoronary and peripheral vein bypass grafts. We investigated the short- and long-term effects of periadventitial application of a sustained-release formulation of rapamycin on experimental pig vein grafts with similar dimensions and kinetics to human saphenous vein bypass grafts. METHODS AND RESULTS: Periadventitial application of rapamycin-eluting polyvinyl alcohol microspheres (60 microg . cm(-2)) to porcine saphenous vein-to-carotid artery interposition grafts inhibited vein graft positive and vascular smooth muscle cell proliferation in 1-week grafts. It also decreased neointima formation and wall thickening in 4-week vein grafts compared with controls. The inhibition of vein graft thickening was not sustained; however, a catch-up phenomenon was observed, and there was no therapeutic benefit evident in 12-week grafts. Increasing the dose of rapamycin to 120 microg . cm(-2) was associated with significant local toxicity manifest by high rates of graft rupture (25%), inhibition of adventitial neoangiogenesis, and a paradoxical acceleration of vein graft disease as evidenced by increased vascular smooth muscle cell proliferation. CONCLUSIONS: Local toxicity and poor long-term efficacy limits the clinical applicability of locally applied, sustained rapamycin release in vein graft disease.

Estrogen attenuates vascular remodeling in Lp(a) transgenic mice.

OBJECTIVE: Although it is well known that Lipoprotein(a) (Lp(a)) is an atherogenic lipoprotein and an independent risk factor for cardiovascular disease, there is no confirmed therapy to decrease Lp(a) or prevent atherosclerosis induced by Lp(a). Thus, it is mandatory to develop novel therapy to prevent atherosclerosis in high Lp(a) concentration. Here, we focused on the effect of estrogen on Lp(a) level and Lp(a)-induced vascular remodeling. METHODS: We employed Lp(a) transgenic mice (human apo(a) yeast artificial chromosome (YAC) and human apoB double transgenic mice). Vascular remodeling was induced by ligation of the common carotid artery and the effect of estrogen was evaluated in female mice after ovariectomy with or without estrogen replacement. RESULTS: Estrogen deficiency caused by ovariectomy increased serum Lp(a), and continuous replacement of 17beta-estradiol (20 microg/kg/day) reversed the change. In the vascular remodeling model induced by carotid artery occlusion, neointima formation was significantly increased in ovariectomized female Lp(a) transgenic mice, but few in male Lp(a) transgenic mice, as compared to wild FVB mice. Importantly, continuous replacement of estrogen in ovariectomized mice significantly attenuated it. In cultured endothelial cells and macrophages, addition of Lp(a) increased mRNA of ICAM-1, VCAM-1, E-selectin and MCP-1 in endothelial cells and TNF-alpha, IL-1beta and MCP-1 in macrophages in a dose-dependent manner. Importantly, pre-treatment with estrogen attenuated these changes in a dose-dependent manner. CONCLUSION: Estrogen negatively regulates both plasma Lp(a) level and Lp(a)-induced vascular remodeling, suggesting that estrogen might be a strong candidate to reduce serum Lp(a) concentration.

Bolus delivery of mesenchymal stem cells to injured vasculature in the rabbit carotid artery produces a dysfunctional endothelium.

Endothelial dysfunction is an important factor in cardiovascular pathology. It has been suggested that pluripotent mesenchymal stem cells (MSCs) may contribute to repair of the endothelium through paracrine pathways. Enhanced re-endothelialization may be associated with a better outcome following angioplasty procedures. We examined the effect of the delivery of MSCs to a denuded vessel in vivo. The right carotid arteries of New Zealand white rabbits were denuded using an uninflated 3-French Fogarty balloon catheter. 1 x 10(5) MSCs in a bolus of 150 microL were then delivered intraluminally and allowed to dwell for 20 min. MSC engraftment was assessed using PKH-26 labeling and transduction with adenoviral reporter genes. Vessels were examined at 2 weeks for levels of endothelialization, as well as for neointimal hyperplasia and vasomotor function. Engraftment of MSCs was noted in the vessel wall following local arterial delivery. Endothelialization was improved following bolus MSC delivery at 2 weeks post-intervention. However, this endothelium is manifestly dysfunctional as indicated by a significant impairment in vasomotor activity and a significant increase in neointimal formation post-bolus delivery. Consistent with the formation of a dysfunctional endothelium, there was a higher rate of vessel occlusions in bolus-treated vessels due to not only predominately thrombosis but also neointimal hyperplasia. Our results suggest that naive MSCs delivered as a bolus to the occluded injured vascular segment generate dysfunctional endothelium presenting a risk of vessel occlusion. Such risks are important and need to be further assessed.

Long-term effects of novel biodegradable, polymer-coated, sirolimus-eluting stents on neointimal formation in a porcine coronary model.

The postulated relationship between nonbiodegradable polymers and late stent thrombosis has led to a concerted effort to seek alternative biodegradable polymers for drug delivery. The purpose of this study was to evaluate the long-term effects of novel sirolimus-eluting stents (SES) with biodegradable polylactic-co-glycolic acid (PLGA) polymer on neointimal thickening in a porcine coronary model. Three types of stents were implanted in different coronary arteries of the same mini-swine: bare cobalt-chromium stents (BMS); PLGA-coated-only stents (PCOS); and PLGA-coated, sirolimus-eluting stents (PCSES). A total of 26 animals underwent successful placement of 78 oversized stents (each stent-group, n = 26) in the coronary arteries with histopathologic analysis and Western blot at 28 days, 3 months, or 1 year. At 28 days and 3 months, the mean neointimal area was about two-fold lower in PCSES versus BMS or PCOS. At 1 year, the mean intimal area was similar for PCSES (1.76 +/- 0.28 mm(2)) and BMS (2.06 +/- 0.23 mm(2), P = 0.051). Western blot analysis demonstrated decreased expression of p27(kip1) in the vessel wall 3 months after PCSES implantation as compared with 28 days. PCSES effectively reduced in-stent neointimal formation for the first 3 months in this porcine coronary model. Beyond 3 months, neointimal proliferation was not substantially inhibited by PCSES. The observed delayed neointimal hyperplasia with PCSES may be partly related to the potential side effects of sirolimus and/or late insufficient arterial drug levels.

Smooth muscle Notch1 mediates neointimal formation after vascular injury.

BACKGROUND: Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. METHODS AND RESULTS: We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared with wild-type or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3-/- mice. Indeed, SMCs derived from explanted aortas of either N1(+/-)- or smN1+/- mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3-/- mice. This correlated with decreased staining of proliferating cell nuclear antigen-positive cells and increased staining of cleaved caspase-3 in the intima of N1(+/-)- or smN1+/- mice. In SMCs derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. CONCLUSIONS: These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases.