Identification of Three Cuproptosis-specific Expressed Genes as Diagnostic Biomarkers and Therapeutic Targets for Atherosclerosis.

Atherosclerosis is a chronic, inflammatory disease characterized by a lipid-driven infiltration of inflammatory cells in large and medium arteries and is considered to be a major underlying cause of cardiovascular diseases. Cuproptosis, a novel form of cell death, is highly linked to mitochondrial metabolism and mediated by protein lipoylation. However, the clinical implication of cuproptosis-related genes (CRGs) in atherosclerosis remains unclear. In this study, genes collected from the GEO database intersected with CRGs were identified in atherosclerosis. GSEA, GO and KEGG pathway enrichment analyses were performed for functional annotation. Through the random forest algorithm and the construction of a protein-protein interaction (PPI) network, eight selected genes (LOXL2, SLC31A1, ATP7A, SLC31A2, COA6, UBE2D1, CP and SOD1) and a vital cuproptosis-related gene FDX1 were then further validated. Two independent datasets (GSE28829 (N = 29), GSE100927 (N = 104)) were collected to construct the signature of CRGs for validation in atherosclerosis. Consistently, the atherosclerosis plaques showed significantly higher expression of SLC31A1, SLC31A2 and lower expression of SOD1 than the normal intimae. The area under the curve (AUC) of SLC31A1, SLC31A2 and SOD1 performed well for the diagnostic validation in the two datasets. In conclusion, the cuproptosis-related gene signature could serve as a potential diagnostic biomarker for atherosclerosis and may offer novel insights into the treatment of cardiovascular diseases. Based on the hub genes, a competing endogenous RNA (ceRNA) network of lncRNA-miRNA-mRNA and a transcription factor regulation network were ultimately constructed to explore the possible regulatory mechanism in atherosclerosis.

Development of Albumin Coupled, Cholesterol Stabilized, Lipid Nanoemulsion of Methotrexate, and TNF-α Inhibitor for Improved In Vivo Efficacy Against Rheumatoid Arthritis.

Methotrexate (MTX; an anti-folate) and etanercept (ET; a TNF-α inhibitor) are used against arthritis; however, limitations like short biological half-life, low cutaneous absorption, and acidic instability limit their clinical relevance. Therefore, the aim of the investigation was to develop albumin coupled lipid nanoemulsion of MTX and ET for improved efficacy by virtue of their controlled release and specificity at the arthritic site. This emulsion was prepared by high-speed homogenization and stabilized using cholesterol. Lipid nanoemulsion of MTX and ET (MTX+ET-LNE) was coupled with albumin (MTX+ET-ALNE). MTX+ET-ALNE was characterized on the basis of particle size (410 ± 25.4 nm), PDI (0.160), and zeta potential (+38.6 ± 5.6 mV) and evaluated for pH (6.15), drug content (97.7 ± 2.17%), entrapment efficiency (76 ± 4.6%), in vitro release, and in vitro cytotoxicity. About 82.6 ± 9.60% release of MTX+ET was observed in 24 h from the developed MTX+ET-ALNE which may help maintain therapeutic level of drugs in blood at least for one day. No toxicity was observed when Raw 264.7 cells were treated with MTX+ET-ALNE, and no causalities of mice were observed at experimental in vivo dose (10 mg/kg BW) of MTX+ET in MTX+ET-ALNE-treated group. MTX+ET-ALNE treatment has alleviated arthritic scores and inflammatory cytokines level in a very significant manner when compared with MTX+ET-LNE and MTX+ET solutions. MTX+ET-ALNE-treated group restored histological alterations (cartilage/bone erosion, inflammatory cell infiltration, synovial hyperplasia, and narrower joint space) as observed in diseased treated groups. In conclusion, MTX+ET-ALNE can be opted as efficacious and clinically pertinent option to the current medication systems of arthritis.

Regulation of microRNA-155 in endothelial inflammation by targeting nuclear factor (NF)-κB P65.

Increasing evidences have illuminated the fundamental role of inflammation in mediating all stages of atherosclerosis. miR-155, a typical multi-functional miRNA, has recently emerged as a novel component of inflammatory signal transduction in the pathogenesis of atherosclerosis. However, little is known about whether endothelial highly expressed miR-155 can regulate endothelial inflammation-related transcription factors and the predicted role of miR-155 as a negative feedback regulator in endothelial inflammation involved in atherosclerosis. Bioinformatics analysis showed that RELA (nuclear factor-κB p65) is a potential target gene of miR-155 and this was confirmed by a luciferase reporter assay. Our results show that microRNA-155 mediate endothelial inflammation and decrease NFкB p65 and adhesion molecule expression in TNFα-stimulated endothelial cells. Transfection with miR-155 significantly inhibited TNFα-induced monocyte adhesion to endothelium. Inhibition of miR-155 enhanced p65 level and endothelial inflammatory response which was counteracted through the depletion of P65 by Si-P65. On the other hand, knockdown of eNOS, another target of miR-155, while transfecting with miR-155 inhibitor resulted in more significant inflammatory response. miR-155 is highly expressed in TNFα treated HUVECs, deprived of endogenous p65 could reverse TNFα-induced upregulation of miR-155. Thus, TNFα induced miR-155 may serve as a negative feedback regulator in endothelial inflammation involved in atherosclerosis by targeting nuclear transcription factor P65. These results provide a rationale for intervention of intracellular microRNA as possible anti-atherosclerotic targets.

A numerical study on the siphonic effect of enhanced external counterpulsation at lower extremities with a coupled 0D-1D closed-loop personalized hemodynamics model.

Enhanced external counterpulsation (EECP) is a treatment and rehabilitation approach for ischemic diseases, including coronary artery disease. Its therapeutic benefits are primarily attributed to the improved blood circulation achieved through sequential mechanical compression of the lower extremities. However, despite the crucial role that hemodynamic effects in the lower extremity arteries play in determining the effectiveness of EECP treatment, most studies have focused on the diastole phase and ignored the systolic phase. In the present study, a novel siphon model (SM) was developed to investigate the interdependence of several hemodynamic parameters, including pulse wave velocity, femoral flow rate, the operation pressure of cuffs, and the mean blood flow changes in the femoral artery throughout EECP therapy. To verify the accuracy of the SM, we coupled the predicted afterload in the lower extremity arteries during deflation using SM with the 0D-1D patient-specific model. Finally, the simulation results were compared with clinical measurements obtained during EECP therapy to verify the applicability and accuracy of the SM, as well as the coupling method. The precision and reliability of the previously developed personalized approach were further affirmed in this study. The average waveform similarity coefficient between the simulation results and the clinical measurements during the rest state exceeded 90%. This work has the potential to enhance our understanding of the hemodynamic mechanisms involved in EECP treatment and provide valuable insights for clinical decision-making.

Hemodynamics of ventricular-arterial coupling under enhanced external counterpulsation: An optimized dual-source lumped parameter model.

BACKGROUND AND OBJECTIVE: Enhanced external counterpulsation (EECP) is a mechanically assisted circulation technique widely used in the rehabilitation and management of ischemic cardiovascular diseases. It contributes to cardiovascular functions by regulating the afterload of ventricle to improve hemodynamic effects, including increased diastolic blood pressure at aortic root, increased cardiac output and enhanced blood perfusion to multiple organs including coronary circulation. However, the effects of EECP on the coupling of the ventricle and the arterial system, termed ventricular-arterial coupling (VAC), remain elusive. We aimed to investigate the acute effect of EECP on the dynamic interaction between the left ventricle and its afterload of the arterial system from the perspective of ventricular output work. METHODS: A neural network assisted optimization algorithm was proposed to identify the ordinary differential equation (ODE) relation between aortic root blood pressure and flow rate. Based on the optimized order of ODE, a lumped parameter model (LPM) under EECP was developed taking into consideration of the simultaneous action of cardiac and EECP pressure sources. The ventricular output work, in terms of aortic pressure and flow rate cooperated with the LPM, was used to characterize the VAC of ventricle and its afterload. The VAC subjected to the principle of minimal ventricular output work was validated by solving the Euler-Poisson equation of cost function, ultimately determining the waveforms of aortic pressure and flow rate. RESULTS: A third-order ODE can precisely describe the hemodynamic relationship between aortic pressure and flow rate. An optimized dual-source LPM with three energy-storage elements has been constructed, showing the potential in probing VAC under EECP. The LPM simulation results demonstrated that the VAC in terms of aortic pressure and flow rate yielded to the minimal ventricular output work under different EECP pressures. CONCLUSIONS: The ventricular-arterial coupling under EECP is subjected to the minimal ventricular output work, which can serve as a criterion for determining aortic pressure and flow rate. This study provides insight for the understanding of VAC and has the potential in characterizing the performance of the ventricular and arterial system under EECP.

Early enhanced external counter pulsation improves neurological recovery after the return of spontaneous circulation in a mongrel dog cardiac arrest model*.

OBJECTIVES: The aim of this study was to investigate whether early enhanced external counter pulsation therapy after cardiopulmonary resuscitation improved neurological outcome in a mongrel dog cardiac arrest model. DESIGN: Randomized, animal study. SETTING: Assisted circulation laboratory. SUBJECTS: Twenty-four healthy male adult dogs (12-14 kg). INTERVENTIONS: After minutes of untreated ventricular fibrillation followed by 2 minutes of cardiopulmonary resuscitation, the dogs were randomized to receive 4 hours of enhanced external counter pulsation therapy, to receive 4 hours of hypertension with over 140 mm Hg or to be a control. MEASUREMENTS: Blood pressure and left ventricular ejection fraction were recorded. Cerebral flow was assessed using magnetic resonance imaging. Arterial blood gases and endothelium-derived vasoactive substances were assessed before cardiac arrest and 4 hours after the return of spontaneous circulation. Neurological outcome was assessed by the neurologic deficit score and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. RESULTS: Enhanced external counter pulsation significantly improved the left ventricular ejection fraction and increased common carotid artery blood flow and shear stress. Enhanced external counter pulsation increased both relative cerebral blood volume (RCBV, p = 0.043) and relative cerebral blood flow (RCBF, p = 0.012) in animals 4 hours after return of spontaneous circulation. Enhanced external counter pulsation therapy promoted the production of nitric oxide and tissue plasminogen activator and decreased the release of endothelin-1 (p = 0.013) after return of spontaneous circulation. Treatment with norepinephrine in the high mean artery pressure also increased common carotid artery blood flow and shear stress. However, no effects on the left ventricular ejection fraction, the production of nitric oxide and tissue plasminogen activator, or the release of endothelin-1 were found. The neurologic deficit scores of the animals were significantly lower at 24, 48, 72, and 96 hours in the enhanced external counter pulsation group, as well as at 24, 72, and 96 hours compared with animals in the control group after return of spontaneous circulation. Fewer apoptotic neurons were observed in the animals in the enhanced external counter pulsation group compared with the animals in the control and hypertension groups. CONCLUSIONS: These data indicated that the treatment of early enhanced external counter pulsation improved neurological outcome by both increasing cerebral blood flow and improving the recovery of microcirculation after return of spontaneous circulation. The treatment of early enhanced external counter pulsation can be a good option for protecting the brain after return of spontaneous circulation.

Enhanced external counterpulsation improves cerebral blood flow following cardiopulmonary resuscitation.

BACKGROUND: To investigate the therapeutic value of enhanced external counterpulsation (EECP) on recovery of cerebral blood flow following cardiac arrest (CA) and successful resumption of spontaneous circulation (ROSC) by cardiopulmonary resuscitation. METHODS: CA models were conducted using beagle dogs induced by alternating current. After successful ROSC by cardiopulmonary resuscitation, 16 dogs were randomly divided into the EECP and control group (n = 8 per group). Dogs underwent dynamic contrast-enhanced and diffusion-weighted magnetic resonance imaging at baseline prior to CA and during the 3 days following ROSC. Mean blood pressure, right common carotid artery blood flow, intracranial microcirculation and blood lactate levels were measured. Neurological outcome was assessed by the neurologic deficit score. Hematoxylin-eosin staining and transmission electron microscopy were performed for morphology and microconstruction of the cerebral cortex. RESULTS: The EECP group exhibited a significant elevation in right common carotid artery blood flow, intracranial microcirculation and a substantial decrease in blood lactate levels relative to the control group. Relative cerebral blood flow and volume were higher in the EECP group during the 3 days. Apparent diffusion coefficients were significantly higher in the EECP group on the first and third days. After ROSC, the neurologic deficit score was significantly higher in the control group compared to those in the EECP group during the three days of experiment. The cell swelling of neurons and increase of mitochondrial mass were more pronounced in the control group. CONCLUSION: EECP is beneficial for recovery of cerebral blood flow and attenuation of ischemic cerebral edema following CA and successful ROSC.

Expanded human cord blood-derived endothelial progenitor cells salvage infarcted myocardium in rats with acute myocardial infarction.

1. Cell transplantation has promise as a therapeutic option for restoring impaired heart function after acute myocardial infarction (AMI). However, the optimal cell type to use remains controversial. We investigated the therapeutic efficacy and feasibility of intramyocardial transplantation of human umbilical cord blood-derived endothelial progenitor cells (hUCB-EPC) in rats with AMI. 2. The Wistar rats myocardial infarction model was established by ligating the left anterior descending artery. The labelled hUCB-EPC were transplanted through intramyocardial injection. Left ventricular function was assessed using a pressure-volume catheter and echocardiogram. Anti-VIII immunohistochemistry staining was used to reflect the degree of angiogenesis in peri-infarcted areas by calculating the average capillary density. The fibrosis degree of infarcted myocardium was analysed by Masson staining and the collagen volume fraction was calculated. 3. The labelled donor endothelial progenitor cells were detected in the new microvessels in host myocardium by double-positive staining with CM-Dil and FITC-UEA-l. An increase in left ventricular ejection fraction, left ventricular fractional shortening, left ventricular end-systolic pressure, first derivative of left ventricular pressure (+dP/dtmax and -dP/dtmax), as well as a decrease in the left ventricular end-diastolic pressure in rats with cell therapy indicated a significant improvement in global heart function. The cell therapy group had increased microvessel formation and a decreased degree of myocardial fibrosis compared to the control group. Moreover, the degree of myocardial fibrosis was less than that of the control group. 4. The improved global heart function and decreased cardiac fibrosis in rats with AMI implies the potential benefit of hUCB-EPC transplantation.

Investigation of the relationship between venticular fibrillation duration and cardiac/neurological damage in a rabbit model of electrically induced arrhythmia.

BACKGROUND: To establish a simple, economic, and reliable alternating current (AC)-induced cardiac arrest (ACCA) model in rabbits for cardiopulmonary cerebral resuscitation research. METHODS: Ventricular fibrillation was induced in 27 New Zealand rabbits by external transthoracic AC, which were randomly divided into three groups according to the duration of untreated ACCA (ACCA-3 minutes, ACCA-5 minutes, and ACCA-8 minutes). After ACCA, all animals received cardiopulmonary resuscitation for 2 minutes and subsequent defibrillation until return of spontaneous circulation (ROSC). The troponin I levels were measured at 4 hours after ROSC. Animals died spontaneously or were killed at 72 hours after ROSC. The hippocampus were removed and fixed in 3% formalin. TdT-mediated dUTP-biotin nick end labeling and Nissl stainings were performed in 10-µm thickness coronal sections. Furthermore, two rabbits (without induction of ventricular fibrillation, cardiopulmonary resuscitation, and defibrillation) served as normal control group. RESULTS: Mean survival times after ROSC were 48.57 hours ± 24.70 hours, 18.0 hours ± 15.13 hours, and 3.88 hours ± 2.39 hours for groups ACCA-3 minutes, ACCA-5 minutes, and ACCA-8 minutes, respectively. Survival was significantly different between ACCA-3 minutes and other two groups (p = 0.002 and p = 0.01). Neuronal necrosis and apoptosis were found in the hippocampus CA1, CA2, and CA3 areas of group ACCA-3 minutes. In contrast, neuronal necrosis and TdT-mediated dUTP-biotin nick end labeling positive cells were fewer in control animals. CONCLUSIONS: The rabbits in group ACCA-3 minutes had significant neuronal damage with apoptosis in hippocampus CA1, CA2, and CA3 areas at 72 hours after ROSC and survived longer than those in other groups. The model we describe may be a simple, economic, and reliable model for experimental investigation on cardiopulmonary cerebral resuscitation.

[Effect of external counterpulsation on morphological damage of endomembrane and endothelium-dependent vasodilatation in the carotid arteries in hypercholesterolemic pigs].

OBJECTIVE: To explore the effect of long-term enhanced external counterpulsation (EECP) on morphological damage of endomembrane and endothelium-dependent vasodilatation of the carotid arteries of hypercholesterolemic pigs. METHODS: Eighteen male infant pigs were randomly divided into three groups according to the contents of their diet: the normal control group (n=6), the high-cholesterol feeding control group (n=6) and EECP group (n=6). Porcine model of hypercholesterolemia was reproduced by feeding animals with high-cholesterol diet. After completion of EECP treatment for 36 hours in EECP group, carotid arterial rings were harvested from all animals. Both scanning and transmission electron microscopy was employed to observe the changes in morphology of their endomembrane. At the same time, their vasodilatation response to variant dose of acetylcholine (Ach) was detected. RESULTS: The surface of endothelium in the normal control group was smooth, and endothelial cells were in regular streamline array, and they were almost in same size, attaching closely to matrix without smooth muscle cell proliferation and lipid infiltration in intima. In contrast, the endothelial cells of hypercholesterolemic pigs were in irregular array, with marked desquamation, resulting in loose linkage. Smooth muscle cells were found to invade into intimal layer and proliferated, and foam cells could also be found in intimal layer. In hypercholesterolemic pigs treated with EECP, slight intimal damage was found. In addition, with Ach dose of 10(-8) mol/L to 10(-5)mol/L, endothelium-dependent vasodilatation ratio in hypercholesterolemic pigs with or without EECP treatment, was significantly lower than that of the normal control group (all P<0.05). However, endothelium-dependent vasodilatation ratio in pigs with EECP treatment was obviously higher compared with hypercholesterolemic pigs without EECP treatment with the dosage of Ach concentration ranged from 10(-7) mol/L to 10(-5) mol/L (all P<0.05). CONCLUSION: Long-term EECP ameliorates both the morphological damage and the impaired endothelium-dependent vasodilatation function resulting from hypercholesterolemia, contributing to prevention of atherosclerosis.

Effect of external counterpulsation on plasma nitric oxide and endothelin-1 levels.

Enhanced external counterpulsation (EECP) significantly augments diastolic blood flow and has been postulated to improve endothelial function by increased shear stress. We examined the effects of EECP on plasma nitric oxide and endothelin-1 (ET-1) levels. Plasma nitrate and nitrite (NOx) and ET-1 levels were measured serially in 13 patients with coronary artery disease who received 1-hour daily treatments of EECP over 6 weeks. During the course of EECP therapy, plasma NOx progressively increased and plasma ET-1 progressively decreased. After 36 hours of EECP, there was a 62 +/- 17% increase in plasma NOx compared with baseline (43.6 +/- 4.3 vs 27.1 +/- 2.6 micromol/L, p <0.0001) and a 36 +/- 8% decrease in plasma ET-1 (76.7 +/- 9.5 vs 119.5 +/- 8.5 pg/L, p <0.0001). At 3 months after completion of EECP, NOx remained 12 +/- 11% above baseline (p = 0.002), and ET-1 remained 11 +/- 10% below baseline (p = 0.0068). Our data provides neurohormonal evidence to support the hypothesis that EECP improves endothelial function.

Transplanted human umbilical cord blood mononuclear cells improve left ventricular function through angiogenesis in myocardial infarction.

BACKGROUND: Human umbilical cord blood contains an abundance of immature stem/progenitor cells, which may participate in the repair of hearts that have been damaged by myocardial infarction (MI). This study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (hUCBC) transplantation on cardiac function and left ventricular remodeling in rat model of MI. METHODS: Forty-five male Wistar rats were randomized into three groups: MI or control group (n = 15), MI plus cell transplantation (n = 15), and sham group (n = 15). Acute myocardial infarction (AMI) was established by ligating the left anterior descending artery, thereafter, hUCBC were implanted into the marginal area of infarcted myocardium. In MI/control group, DMEM was injected instead of hUCBC following the same protocol. Left ventricular function assessment was carried out by echocardiography and invasive hemodynamic measurements one month post MI. All rats were sacrificed for histological and immunochemical examinations. RESULTS: The transplanted hUCBC survived and engaged in the process of myocardial repair in the host heart. Echocardiography demonstrated that left ventricular function improved significantly in the rats that underwent cell transplantation. Hemodynamic studies found a significantly decreased left ventricular end-diastolic pressure (LVEDP) [(21.08 +/- 8.10) mmHg vs (30.82 +/- 9.59) mmHg, P < 0.05], increase in +dp/dt(max) [(4.29 +/- 1.27) mmHg/ms vs (3.24 +/- 0.75) mmHg/ms, P < 0.05), and increase in -dp/dt(max) [(3.71 +/- 0.79) mmHg/ms vs (3.00 +/- 0.49) mmHg/ms, P < 0.05] among MI group with hUCBC transplantation when compared with MI/control group. Masson's trichrome staining revealed that the collagen density in the left ventricle was significantly lower in rats of transplantation group than that in the MI control groups [(6.33 +/- 2.69)% vs (11.10 +/- 3.75)%, P < 0.01]. Based on immunostaining of alpha-actin, the numbers of microvessels were significantly (P < 0.01) increased at the boundary of infarction site. Similarly higher mRNA expression of vascular endothelial growth factor (VEGF) 164 and VEGF188 were found at 7- and 28-day post cell transplantation in MI group with hUCBC transplantation when compared with MI/control group. CONCLUSIONS: Transplanted hUCBC can survive in host myocardium without immunorejection, significantly improve left ventricular remodeling after AMI and promote a higher level of angiogenesis in the infarct zones. All these factors beneficially affect cardiac repair in the setting of MI. Therefore human umbilical cord blood may be potential source for cell-based therapy for AMI.

[Effects of enhanced external counterpulsation in atherosclerosis and NF-kappaB expression: a pig model with hypercholesterolemia].

OBJECTIVE: To study the effects of enhanced external counterpulsation (EECP) on the vascular morphology, and endothelial function using experimentally induced hypercholesterolemic pigs. METHODS: Thirty five male pigs were randomly divided into three groups: 7 normal control animals, 11 hypercholesterolemic animals, and 17 hypercholesterolemic animals receiving EECP. Serum cholesterol was measured. The coronary arteries and aortas were sampled for histopathologic and ultrastructural examination. The NF-kappaB protein expression of porcine coronary arteries was investigated by immunofluorescence. RESULTS: Compared with the normal controls, serum cholesterol levels were significantly higher in the hypercholesterolemic animals with or without EECP. The plaque/intimal area ratio of the aorta decreased significantly in animals receiving EECP [(3.33 +/- 2.40)%, versus (12.03 +/- 7.12)% in those without EECP, P < 0.05]. Lipid deposition, endothelial damage and proliferation of smooth muscle cells were less severe in animals receiving EECP than those not. Moreover, activation and expression of NF-kappaB also decreased significantly (P < 0.05) in animals receiving EECP. CONCLUSIONS: EECP improves the morphology and function of vascular endothelium, and retards the development and progression of atherosclerosis, likely through the inhibition of NF-kappaB signaling pathway.

[Beneficial effects of human umbilical cord blood mononuclear cells transplantation on rats with experimental myocardial infarction].

OBJECTIVE: Human umbilical cord blood contains abundant immature stem/progenitor cells, which may contribute to the repair of infarcted myocardium. Present study aimed to explore the feasibility and effects of human umbilical cord blood mononuclear cells (HUCBC) transplantation for the treatment of myocardial infarction. METHODS: Forty-five male Wistar rats were randomly divided into three groups: (1) Myocardial infarction (MI plus vehicle, n = 15), (2) MI plus cell transplantation (HUCBC were implanted into the peri-infarct area immediately after MI, n = 15), (3) Normal control group (n = 15). After echocardiography and hemodynamic measurements, the rats were sacrificed for histological and immunochemical examinations one month post MI. RESULTS: The transplanted HUCBC survived and participated the repair process in host heart. Significantly improved left ventricular function was evidenced by echocardiography in cell transplantation group compared to the MI control group. Left ventricular end-diastolic pressure was significantly reduced LVEDP (21.08 +/- 8.10) vs (30.82 +/- 9.59) mm Hg, P < 0.05], +dp/dt(max) [(4.29 +/- 1.27) vs (3.24 +/- 0.75) mm Hg/ms, P < 0.05] and -dp/dt(max) increased [(3.71 +/- 0.79) vs (3.00 +/- 0.49) mm Hg/ms, P < 0.05] in cell transplantation rats compared with MI control rats. vWF immunostaining examination showed significantly increased microvessels within the boundary of infarcted myocardium in cell transplantation group compared to the MI control group (P < 0.01). CONCLUSIONS: HUCBC transplantation may improve cardiac function in MI rats by promoting microvessel formation.

Inhibition of the cardiac angiogenic response to surgical FGF-2 therapy in a Swine endothelial dysfunction model.

BACKGROUND: Discrepancy exists between the potent effects of therapeutic angiogenesis in laboratory animals and the marginal results observed in patients with advanced coronary artery disease. In vitro and small animal data suggest that angiogenesis may depend on locally available nitric oxide (NO), but the impact of endothelial dysfunction on therapeutic angiogenesis in the myocardium has been unclear. We compared the effects of clinically applicable angiogenesis methods in swine in which endothelial dysfunction was experimentally induced to that observed in normal swine. METHODS AND RESULTS: Miniswine were fed either a regular (N=13) or hypercholesterolemic diet (N=13) for 20 weeks. Hypercholesterolemic swine showed coronary endothelial dysfunction on videomicroscopy. Animals from both groups received 100 microg of perivascular sustained-release fibroblast growth factor (FGF)-2 in the lateral myocardial territory, previously made ischemic by placement of an ameroid constrictor around the circumflex artery. After 4 weeks of FGF-2 therapy, lateral myocardial perfusion was significantly lower in hypercholesterolemic than in normocholesterolemic swine, both at rest and during pacing (0.44+/-0.04 versus 0.81+/-0.15 mL/min/g at rest, respectively; P=0.006; and 0.50+/-0.06 versus 0.71+/-0.10 mL/min/g during pacing; P=0.02). Hypercholesterolemic swine showed no net increase in perfusion from FGF-2 treatment. Endothelial cell density and FGF receptor-1 expression were significantly lower in the lateral territory of hypercholesterolemic versus normocholesterolemic animals. CONCLUSIONS: The cardiac angiogenic response to FGF-2 treatment using clinically applicable methods was markedly inhibited in hypercholesterolemic swine with coronary endothelial dysfunction. These findings suggest that coronary endothelial dysfunction is major obstacle to the efficacy of clinical angiogenesis protocols and constitutes a target toward making angiogenesis more effective in patients with advanced coronary disease.

Microvessel angiogenesis: a possible cardioprotective mechanism of external counterpulsation for canine myocardial infarction.

BACKGROUND: Enhanced external counterpulsation (EECP) has been demonstrated to be effective in the treatment of patients with coronary artery disease (CAD). It has been proposed that the beneficial effects of EECP observed in clinical studies may be due to the formation of new blood vessels (angiogenesis) and collateral development. However, there is a relative paucity of basic studies to support the proposed mechanisms. METHODS: Twelve Beagle dogs were anesthetized with 3% sodium pentobarbital, 1 mg/kg intraperitoneal injection and mechanically ventilated for the development of myocardial infarction. After coronary occlusion, all animals were randomly assigned to either EECP or control. EECP was given one hour per day, 5 days a week, for a total of 28 to 30 hours treatment over a 6-week course. Immunohistochemical studies of alpha-actin and von Willebrand factor (vWF) were used to detect newly developed microvessels. Systemic and local vascular endothelial growth factor (VEGF) were identified by enzyme linked immunosorbent assay (ELISA) and reverse-transcriptional polymerase chain reaction (RT-PCR) analysis. RESULTS: There was a significant increase in the density of microvessels per mm(2) in the infarcted regions of EECP group compared to control group (vWF, 15.2 +/- 6.3 versus 4.9 +/- 2.1, P < 0.05; alpha-actin, 11.8 +/- 5.3 versus 3.4 +/- 1.2, P < 0.05), along with significant increase of positive vWF and alpha-actin stained area. Both immunohistochemical staining and RT-PCR analysis documented a significant increase in VEGF expression. These factors associated with angiogenesis corresponded to improved myocardial perfusion by 99mTc-sestamibi single-photon emission computed tomography. CONCLUSION: Microvessel angiogenesis may be a mechanism of action for the improved myocardial perfusion after EECP therapy.

Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia.

OBJECTIVES: Enhanced external counterpulsation (EECP) could improve endothelium-dependent vasodilatation of carotid artery and restore imbalance of nitric oxide and endothein-1 in patients with coronary artery disease. Our study was designed to test the hypothesis that long-term EECP may protect vascular endothelial cells from apoptosis by modifying apoptosis-related gene expression. METHODS: Eighteen male Yorkshire pigs were randomly assigned to three groups: usual diet (Normal), high cholesterol diet (HC) and high cholesterol diet plus EECP (HC+EECP). Vascular endothelial cells were isolated from the aortic endothelium and identified by CD31 staining and DiI-Ac-LDL reaction. Morphological changes were observed by both scanning and transmission electronic microscopes. TUNEL technique was applied to detect the apoptotic index of vascular endothelial cells. Two genes, Apaf-1 and BIRC2, were chosen for exploring the potential mechanisms of action at the molecular level. RESULTS: EECP brought a certain degree of alleviation from ultrastructural changes such as shrinking and blebbing of cytomembrane, marginalization, degeneration, and fragmentation of the nucleus. EECP also significantly reduced apoptotic indices while compared with that of control (177±12‰ vs. 237±23‰, P<0.05). The Apaf-1 expression at both protein and mRNA level in pigs of HC+EECP group was significantly decreased than those of the HC group (P<0.05), whereas the BIRC2 expression was significantly enhanced after EECP treatment, documented by immunostaining and semi-quantitative RT-PCR analysis, respectively (P<0.05). CONCLUSIONS: EECP could protect vascular endothelial cells from apoptosis, thereby delaying the progression of early atherosclerotic lesions possibly through transcriptional down-regulation of pro-apoptotic gene Apaf-1, and up-regulation of anti-apoptotic gene BIRC2.

Vasculoprotective properties of enhanced external counterpulsation for coronary artery disease: beyond the hemodynamics.

A growing pool of evidence has shown that enhanced external counterpulsation (EECP) is a non-invasive, safe, low-cost, and highly beneficial therapy for patients with coronary artery disease. However, the exact mechanisms of benefit exerted by EECP therapy remain only partially understood. The favorable hemodynamic effects of EECP were previously considered as the primary mechanism of action. Nevertheless, recent advances have shed light on the shear stress-increasing effects of EECP which are vasculoprotective and anti-atherosclerotic. EECP-induced endothelial shear stress increase may lead to improvement in endothelial function and morphology, attenuation of oxidative stress and inflammation, and promotion of angiogenesis and vasculogenesis. This review summarizes evidence of the potential mechanisms contributing to the immediate and long-term benefits of EECP, from the perspective of its shear stress-increasing effects.

Transplantation of human umbilical cord-derived endothelial progenitor cells promotes re-endothelialization of the injured carotid artery after balloon injury in New Zealand white rabbits.

BACKGROUND: Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI). The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries. METHODS: Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofluorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group (n = 16), while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n = 16). The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemistry and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-ß1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Fluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P < 0.05). von Willebrand factor (vWF) immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.75 ± 2.92 vs. 4.50 ± 1.77, P < 0.05). Compared with the control group, the transplanted group had lower expression of PCNA mRNA (0.67 ± 0.11 vs. 1.25 ± 0.40, P < 0.01) and higher expression of TGF-ß1 mRNA (1.10 ± 0.21 vs. 0.82 ± 0.07, P < 0.05). CONCLUSIONS: EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization.

The changes of brain water diffusion and blood flow on diffusion-weighted and perfusion-weighted imaging in a canine model of cardiac arrest.

OBJECTIVE: To study the changes of brain water diffusion and cerebral haemodynamics of cortical areas using magnetic resonance imaging (MRI) in canine models of cardiac arrest (CA) and restoration of spontaneous circulation (ROSC). The secondary study objective was to evaluate whether MRI can be used to observe haemodynamic disorders in brain microcirculation. METHODS: CA was induced in six beagle dogs using electrical stimulation followed by resuscitation to spontaneous circulation 3 min later. The dogs were scanned using MRI for echo planar, diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI) with injection of Gd-diethylene triamine pentaacetic acid (DTPA) prior to induction of CA and 3 days after ROSC. The arterial blood pressure, unilateral common carotid artery flow and intracranial microcirculation were recorded. RESULTS: All dogs successfully underwent electric-induced ventricular fibrillation which lasted 3 min and were resuscitated to maintain blood pressure stability. Serial MRI scans found that cerebral blood flow (RCBF) decreased in day 1 after ROSC and returned to baseline on day 3. Apparent diffusion coefficient (ADC), however, decreased on day 1 and remained lower than baseline on day 3, with 765.8±82.5×10(-6) mm(2) s(-1) on day 1 and 770.4±59.4×10(-6) mm(2) s(-1) on day 3 comparing to 855.8±43.4×10(-6) mm(2) s(-1) on baseline. CONCLUSIONS: These data provide the evidence that early MRI can be used to observe acute haemodynamic disorders in brain microcirculation in a canine model of cardiac arrest.