Histopathological evaluation of a retinoic acid eluting stent in a rabbit iliac artery model.

This study aimed to evaluate the safety and efficacy of innovative retinoic acid (RA) eluting stents with bioabsorbable polymer. Sixty stents divided in ten groups were implanted in the iliac arteries of 30 rabbits. Two polymers ("A", poly (lactic-co-glycolic acid) and "B", polylactic acid), and three doses ("Low", "Medium" and "High") of RA (groups: AL, AM, AH, BL, BM, BH) were used on cobalt chromium stents (Rontis Corporation), one group of bare stent (C), one group (D) of Everolimus eluting stent (Xience-Pro, Abbot Vascular), and two groups of Rontis Everolimus eluting stents coated with polymer A (EA) and B (EB). Treated arteries were explanted after 4 weeks, processed by methyl methacrylate resin and evaluated by histopathology. None of the implanted stents was related with thrombus formation or extensive inflammation. Image analysis showed limited differences between groups regarding area stenosis (BH, D and EB groups had the lower values). Group BH had lower intimal mean thickness than AH (105.1 vs 75.3 µm, p = 0.024). Stents eluting RA, a non-cytotoxic drug, were not related with thrombus formation and had an acceptable degree of stenosis 4 weeks post implantation. RA dose and type of polymer may play role in the biocompatibility of the stents.

Chronic Empagliflozin Treatment Reduces Myocardial Infarct Size in Nondiabetic Mice Through STAT-3-Mediated Protection on Microvascular Endothelial Cells and Reduction of Oxidative Stress.

Aims: Empagliflozin (EMPA) demonstrates cardioprotective effects on diabetic myocardium but its infarct-sparing effects in normoglycemia remain unspecified. We investigated the acute and chronic effect of EMPA on infarct size after ischemia-reperfusion (I/R) injury and the mechanisms of cardioprotection in nondiabetic mice. Results: Chronic oral administration of EMPA (6 weeks) reduced myocardial infarct size after 30 min/2 h I/R (26.5% ± 3.9% vs 45.8% ± 3.3% in the control group, p < 0.01). Body weight, blood pressure, glucose levels, and cardiac function remained unchanged between groups. Acute administration of EMPA 24 or 4 h before I/R did not affect infarct size. Chronic EMPA treatment led to a significant reduction of oxidative stress biomarkers. STAT-3 (signal transducer and activator of transcription 3) was activated by Y(705) phosphorylation at the 10th minute of R, but it remained unchanged at 2 h of R and in the acute administration protocols. Proteomic analysis was employed to investigate signaling intermediates and revealed that chronic EMPA treatment regulates several pathways at reperfusion, including oxidative stress and integrin-related proteins that were further evaluated. Superoxide dismutase and vascular endothelial growth factor were increased throughout reperfusion. EMPA pretreatment (24 h) increased the viability of human microvascular endothelial cells in normoxia and on 3 h hypoxia/1 h reoxygenation and reduced reactive oxygen species production. In EMPA-treated murine hearts, CD31-/VEGFR2-positive endothelial cells and the pSTAT-3(Y705) signal derived from endothelial cells were boosted at early reperfusion. Innovation: Chronic EMPA administration reduces infarct size in healthy mice via the STAT-3 pathway and increases the survival of endothelial cells. Conclusion: Chronic but not acute administration of EMPA reduces infarct size through STAT-3 activation independently of diabetes mellitus.

Carotid Stent Restenosis and Thrombosis in Rabbits: The Effect of Antiplatelet Agents.

BACKGROUND: The purpose of the study was the comparative assessment of ticagrelor and clopidogrel effects on carotid post-balloon injury (PBI) and on post carotid artery stenting (CAS) rate of in-stent restenosis (ISR) and in-stent thrombosis in atherosclerotic rabbits. METHODS: Forty-eight New Zealand white rabbits on high-fat diet were randomized into 4 groups: A1: PBI and clopidogrel (30 mg/kg/d), A2: PBI and ticagrelor (21 mg/kg twice daily), B1: PBI, CAS, and clopidogrel (30 mg/kg/d), B2: PBI, CAS, and ticagrelor (21 mg/kg twice daily). All rabbits received orally aspirin (10 mg/kg/d) and interventions were performed in their right carotid arteries (RCAs). Optical coherence tomography (OCT) and carotid angiography were performed at end point, while platelet aggregation and lipid profile were measured. After euthanasia both carotids were obtained for histological examination. RESULTS: In B1 group, 3 rabbits presented thrombotic total occlusion of the stents, while none such episode was observed in B2 group. The neointimal areas in RCAs, calculated by OCT, did not differ between A1 and A2 groups, and between B1 and B2 groups (P > .05). From the histological findings, the intima/(media + intima) percentage (%) in RCAs of balloon-injured rabbits did not present any difference between groups (P = .812). Similarly, the immunohistochemically determined accumulation of endothelial cells and macrophages on vascular walls was equivalent between groups (P > .05). CONCLUSION: Following carotid balloon injury and stenting, clopidogrel and ticagrelor did not show any differential effects on the extent of neointimal formation and ISR in atherosclerotic rabbits receiving aspirin. Three thrombotic stent occlusions were noted in the clopidogrel treatment group, but this finding was not statistically significant.

Impaired calcium homeostasis is associated with sudden cardiac death and arrhythmias in a genetic equivalent mouse model of the human HRC-Ser96Ala variant.

AIMS: The histidine-rich calcium-binding protein (HRC) Ser96Ala variant has previously been identified as a potential biomarker for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. Herein, the role of this variant in cardiac pathophysiology is delineated through a novel mouse model, carrying the human mutation in the homologous mouse position. METHODS AND RESULTS: The mouse HRC serine 81, homologous to human HRC serine 96, was mutated to alanine, using knock-in gene targeting. The HRC-Ser81Ala mice presented increased mortality in the absence of structural or histological abnormalities, indicating that early death may be arrhythmia-related. Indeed, under stress-but not baseline-conditions, the HRC-Ser81Ala mice developed ventricular arrhythmias, whilst at the cardiomyocyte level they exhibited increased occurrence of triggered activity. Cardiac contraction was decreased in vivo, ex vivo, and in vitro. Additionally, Ca2+ transients and SR Ca2+ load were both reduced suggesting that cytosolic Ca2+ overload is not the underlying proarrhythmic mechanism. Interestingly, total SR Ca2+ leak was increased in HRC-Ser81Ala cardiomyocytes, without an increase in Ca2+ spark and wave frequency. However, Ca2+ wave propagation was significantly slower and the duration of the associated Na/Ca exchange current was increased. Moreover, action potential duration was also increased. Notably, Ca2+/Calmodulin kinase II (CaMKII) phosphorylation of the ryanodine receptor was increased, whilst KN-93, an inhibitor of CaMKII, reduced the occurrence of arrhythmias. CONCLUSIONS: The homologous mutation Ser81Ala in HRC in mice, corresponding to Ser96Ala in humans, is associated with sudden death and depressed cardiac function. Ventricular arrhythmias are related to abnormal Ca2+ cycling across the SR. The data further support a role for CaMKII with the perspective to treat arrhythmias through CaMKII inhibition.

Effect of ovariectomy and Sideritis euboea extract administration on large artery mechanics, morphology, and structure in middle-aged rats.

BACKGROUND: Arterial function is regulated by estrogen, but no consistent pattern of arterial mechanical remodeling in response to depleted estrogen levels is available. OBJECTIVE: To examine long-term effects of ovariectomy (OVX) on the mechanical properties, morphology, and histological structure of the carotid artery in middle-aged rats and a potentially protective effect of Sideritis euboea extract (SID), commonly consumed as "mountain tea". METHODS: 10-month-old female Wistar rats were allocated into control (sham-operated), OVX, OVX+SID, and OVX+MALT (maltodextrin; excipient used for dilution of SID) groups. They were sacrificed after 6 months and their carotid arteries were submitted to inflation/extension tests and to dimensional and histological evaluation. RESULTS: Remodeling in OVX rats was characterized by a decreased in situ axial extension ratio, along with increased opening angle, thickness, and area of the vessel wall and of its medial layer, but unchanged lumen diameter. Compositional changes involved increased elastin/collagen densities. Characterization by the "four-fiber" microstructure-motivated model revealed similar in situ biaxial response of carotid arteries in OVX and control rats. CONCLUSIONS: Carotid artery remodeling in OVX rats was largely consistent with hypertensive remodeling, despite the minor arterial pressure changes found, and was not altered by administration of SID, despite previous evidence of its osteo-protective effect.

Empagliflozin Limits Myocardial Infarction in Vivo and Cell Death in Vitro: Role of STAT3, Mitochondria, and Redox Aspects.

Empagliflozin (EMPA), a drug approved for type 2 diabetes management, reduced cardiovascular death but is unknown if it reduces myocardial infarction. We sought to investigate: (i) the effect of EMPA on myocardial function and infarct size after ischemia/reperfusion in mice fed with western diet (WD), (ii) the underlying signaling pathways, (iii) its effects on cell survival in rat embryonic-heart-derived cardiomyoblasts (H9C2) and endothelial cells (ECs). To facilitate the aforementioned aims, mice were initially randomized in Control and EMPA groups and were subjected to 30 min ischemia and 2 h reperfusion. EMPA reduced body weight, blood glucose levels, and mean arterial pressure. Cholesterol, triglyceride, and AGEs remained unchanged. Left ventricular fractional shortening was improved (43.97 ± 0.92 vs. 40.75 ± 0.61%) and infarct size reduced (33.2 ± 0.01 vs. 17.6 ± 0.02%). In a second series of experiments, mice were subjected to the above interventions up to the 10th min of reperfusion and myocardial biopsies were obtained for assessment of the signaling cascade. STAT3 was increased in parallel with reduced levels of malondialdehyde (MDA) and reduced expression of myocardial iNOS and interleukin-6. Cell viability and ATP content were increased in H9C2 and in ECs. While, STAT3 phosphorylation is known to bestow infarct sparing properties through interaction with mitochondria, we observed that EMPA did not directly alter the mitochondrial calcium retention capacity (CRC); therefore, its effect in reducing myocardial infarction is STAT3 dependent. In conclusion, EMPA improves myocardial function and reduces infarct size as well as improves redox regulation by decreasing iNOS expression and subsequently lipid peroxidation as shown by its surrogate marker MDA. The mechanisms of action implicate the activation of STAT3 anti-oxidant and anti-inflammatory properties.

Acute histological changes of the lung after experimental Fontan circulation in a swine model.

BACKGROUND: Histological changes of the lungs were studied after the establishment of a modified total cavopulmonary connection (TCPC) without the use of cardiopulmonary bypass (CPB) or other means of temporary bypass on a swine model. MATERIAL/METHODS: 8 open chest-anesthetized pigs Landrace x Large White pigs (mean weight 43kg, mean age 4.5 months) underwent TCPC by the use of an appropriate size Y-shaped conduit connecting the superior and inferior caval veins (end-to-end anastomosis) to the pulmonary trunk (end-to-side anastomosis). After sternotomy, a wedge resection of the lung parenchyma was performed at baseline. Hemodynamic stability was sustained after TCPC establishment and 2 hours later another wedge resection of the lung was performed (from the same anatomic area). Histological studies were conducted by hematoxylin and eosin staining. RESULTS: All samples (n=8) at baseline were consistent with normal lung parenchyma. After the establishment of TCPC, all samples (n=8) revealed moderate mononuclear infiltration adjacent to pulmonary alveoli and bronchioles, findings compatible with bronchiolitis. CONCLUSIONS: In a normal swine model, 2 hours after the establishment of Fontan circulation without the use of CPB, pathologic examination of the lungs revealed bronchiolitis. Further research is needed to clarify these findings and the potential implications to the Fontan circulation, either immediate or long-term.

Time course of flow-induced adaptation of carotid artery biomechanical properties, structure and zero-stress state in the arteriovenous shunt.

Numerous studies have provided evidence of diameter adaptation secondary to flow-overload, but with ambiguous findings vis à vis other morphological parameters and information on the biomechanical aspects of arterial adaptation is rather incomplete. We examined the time course of large-artery biomechanical adaptation elicited by long-term flow-overload in a porcine shunt model between the carotid artery and ipsilateral jugular vein. Post-shunting, the proximal artery flow was doubled and retained so until euthanasia (up to three months post-operatively), without pressure change. This hemodynamic stimulus induced lumen diameter enlargement, accommodated by elastin fragmentation and connective tissue accumulation, as witnessed by optical and confocal microscopy. Heterogeneous mass growth of the adventitia was observed at the expense of the media, associated with declining residual strains and opening angle at three months. The in vitro elastic properties of shunted arteries determined by inflation/extension testing were also modified, with the thickness-pressure curves shifted to larger thicknesses and the diameter-pressure curves shifted to larger diameters at physiologic pressures, resulting in normalization of intramural and shear stresses within fifteen and thirty days, respectively. We infer that the biomechanical adaptation in moderate flow-overload leads to normalization of intimal shear, without, however, restoring compliance and distensibility at mean in vivo pressure to control levels.

The effect of propranolol on aortic structure and function in normotensive rats.

INTRODUCTION: Beta-blocking agents are widely used for the treatment of many cardiovascular diseases. The effect of these agents, however, on the aortic wall structure and function has not been well defined. The present study was undertaken to investigate the effect of therapy with propranolol on wall structure and aortic function in rats. METHODS: 20 healthy Wistar rats (350-400 g) were assigned to a control group (n=8), with rats receiving only water and food, and an experimental group (n=12), in which 100 mg/kg/day propranolol was administered in the drinking water. Three months after initiation of treatment, aortic pressures and aortic pulse wave velocity (PWV) were measured using high-fidelity Millar catheters. Extensive histopathologic studies were performed in the wall of the descending thoracic aorta. RESULTS: Systolic, mean, diastolic, and pulse pressure were significantly lower in the propranolol-treated rats compared to controls (p<0.05). For any given systolic, mean, and pulse pressure, PWV was greater in the propranolol-treated animals (p<0.05). The heart rate was lower and the response to isoproterenol infusion was less in the propranolol-treated animals. Smooth muscle content was decreased and collagen content was increased in the aortic wall of the propranolol-treated animals compared to controls. CONCLUSIONS: Long-term propranolol administration elicits an increase in PWV adjusted for aortic pressure. This may be related to accumulation of collagen in the aortic wall at the expense of smooth muscle cells. The aortic stiffening may explain some of the reported data, suggesting that the effect of ß-blockade therapy in patients with arterial hypertension may be inferior to other pharmacologic agents.

Surgical thoracic sympathectomy induces structural and biomechanical remodeling of the thoracic aorta in a porcine model.

BACKGROUND: Sympathetic innervation exerts marked effects on vascular smooth muscle cells, including a short-term homeostatic (vasoconstrictor) and a direct trophic action promoting differentiation. However, the role of sympathetic nervous system in long-term structural and functional modulation of the aortic wall is yet undefined. METHODS: Six Landrace pigs underwent bilateral thoracic sympathectomy from the stellate to T8 ganglion, whereas 10 pigs underwent sham operation. Animals were sacrificed 3 mo postoperatively. Histometrical examination was performed on specimens from the thoracic (TA) and abdominal aorta (AA) utilizing an image-processing system. A uniaxial tensile tester was utilized for biomechanical evaluation; parameters of extensibility, strength, and stiffness of aortic tissue were calculated. RESULTS: Structural aortic remodeling of sympathectomized animals was observed, including increased inner aortic diameter in TA (15.3 ± 0.4 versus 10.4 ± 0.2 mm, P < 0.001) and AA (6.7 ± 0.3 versus 5.3 ± 0.2 mm, P = 0.002), and increased wall thickness in TA (2.0 ± 0.1 versus 1.6 ± 0.1 mm, P < 0.001) but not AA. Microscopic image analysis revealed increased elastin (TA: 50.1 ± 1.1 versus 29.7% ± 0.6%, P < 0.001; AA: 20.4 ± 2.1 versus 16.3% ± 0.6%, P = 0.03) and collagen density (only in TA: 22.0 ± 0.9 versus 15.4% ± 0.5%, P < 0.001), and decreased smooth muscle density (TA: 27.6 ± 1.3 versus 54.9% ± 0.7%, P < 0.001; AA: 57.2 ± 1.5 versus 63.4% ± 0.8%, P < 0.001). Sophisticated biomechanical analysis demonstrated that following sympathectomy, TA was equally extensible but manifested augmented strength (1344 ± 73 versus 1071 ± 52 kPa, P = 0.004) and stiffness (6738 ± 478 versus 5026 ± 273 kPa, P = 0.003), in accordance with extracellular matrix protein accumulation in that region. Differences in the AA were non-significant. CONCLUSIONS: Chronic thoracic sympathetic denervation causes significant structural and biomechanical remodeling of the thoracic aorta. Possible clinical implications for patients undergoing thoracic sympathectomy or chronically treated with sympathetic blockers require further investigation.

Minimal invasive technique for gene delivery in porcine liver lobe segment.

INTRODUCTION: The feasibility and outcome of large volume injection of gene solution in a segment of a liver lobe, without backflow, were studied in a porcine model, using a custom-designed balloon catheter. METHOD: Eight anesthetized pigs underwent successful injection of 200 ml of gene solution at a rate of 20 ml/s via a minimally invasive technique without backflow. A custom-made balloon catheter was introduced under fluoroscopy guidance into the right lateral liver lobe via the right external jugular vein. The vein of the liver lobe was occluded with the balloon catheter and contrast material was injected to check if total occlusion was achieved. Since there was no backflow an angiographic pump injected the solution. The catheter was left in place for 10 min. Then contrast material was injected to check whether the vein remained occluded. RESULTS: All animals tolerated the procedure without obvious adverse effects. Ultrasound scan showed no gross changes within liver three days following the infusion. A transient rise in platelet count was observed which returned to normal after 13 days and remained stable; all other biochemistry values were normal. CONCLUSIONS: Injecting large volume of gene solution in a liver lobe segment using this minimally invasive technique in a porcine model is possible, making the development of a successful gene transfer protocol in humans feasible.

Experimentally modified Fontan circulation in an adolescent pig model without the use of cardiopulmonary bypass.

BACKGROUND: The feasibility and the hemodynamic outcome of Fontan circulation, without the use of cardiopulmonary bypass, were studied on a beating heart of an adolescent pig model, using a modified total cavopulmonary connection. MATERIAL/METHODS: Eight open-chest anesthetized pigs underwent a successful total cavopulmonary connection with the use of an appropriate Y-shaped Dacron-type conduit. Through a median sternotomy, the distal part of the superior vena cava was anastomosed end-to-end to one side of the conduit. The other side of the graft was anastomosed end-to-side to the main pulmonary artery. The conduit was tailored to an appropriate length and anastomosed end-to-end to the inferior vena cava. The hemodynamic status of the animals was recorded before and after the establishment of the total cavopulmonary connection. RESULTS: Forty-five minutes after completion of total cavopulmonary connection, and for a total of 1 hour, hemodynamic measurements showed a decrease in mean arterial and mean pulmonary artery pressures, heart rate and cardiac output. The inferior vena caval pressure and total pulmonary vascular resistance were increased. CONCLUSIONS: A total cavopulmonary connection, performed on a beating heart, without extracorporeal circulation or other means of temporary bypass, although it is technically demanding, is feasible.

Time-course of mechanical changes of the rat aorta following chronic beta-blocker treatment.

INTRODUCTION: The mechanical properties of the aorta play an important role in arterial homeostasis and constitute a prognostic factor in cardiovascular disease. This study determined the time-course of mechanical changes of the thoracic aorta following prolonged beta (beta)-blocker treatment. METHODS: Sixty-six healthy male Wistar rats were randomized to 4 groups. Group A was divided into subgroups A1 (n=6), A2 (n=6), and A3 (n=6), with animals receiving only water. In groups B (n=16), C (n=16), and D (n=16), propranolol was added to the drinking water (100 mg/kg/day). Animals of groups A1 and B, A2 and C, and A3 and D were sacrificed after 1, 2, and 3 months. The effect of beta-blockade was assessed by heart rate changes in response to isoproterenol infusion. The thoracic aorta was excised and submitted to mechanical testing. Regression analysis was performed to evaluate the relationship between elastic modulus and stress for low (part I), physiologic (part II), and high (part III) stresses. RESULTS: Data from subgroups A1, A2, and A3 were pooled together and were used as a control. Differences were found in the regression parameters of parts II and III between the propranolol-treated groups and controls, indicating that the aorta was stiffer in propranolol-treated rats compared to controls at physiologic stresses, and at physiologic and high strains. Changes developed progressively with the duration of treatment. No differences were found in the regression parameters of part I, indicative of non-varying elastic modulus, i.e. stiffness, at low stresses and strains. CONCLUSIONS: Chronic blockade of beta-adrenergic receptors induces changes in the mechanical properties of the thoracic aorta. Aortic stiffening in response to beta-blocker treatment may be of great clinical significance.

The effects of hypothyroidism on the mechanical properties and histomorphological structure of the thoracic aorta.

This experimental study investigates the effects of hypothyroidism on the descending thoracic aorta. Hypothyroidism was induced in 20 male Wistar rats by administering 0.05% of 6-n propyl 2-thiouracil (PTU) in their drinking water for 8 weeks. Euthyroid rats were used as controls. Animals were sacrificed and longitudinal strips of the descending aorta were subjected to various preselected levels of stress in a uniaxial tensile-testing device. Analysis of stress-strain, elastic modulus-strain curves disclosed significant differences between groups, indicative of stiffer aortas in hypothyroid animals at the upper physiologic and higher levels of pressure. Remodeling of the aortic wall of hypothyroid animals revealed significant histological changes. The thoracic aorta of hypothyroid rats compared with that of euthyroid ones became stiffer at high strains, including the upper physiologic range, loosing part of its distensibility. Hypothyroidism was also associated with diameter enlargement and substantial lengthening of the aorta.

The mechanical performance and histomorphological structure of the descending aorta in hyperthyroidism.

Thyroid hormones decrease systemic vascular resistance by directly affecting vascular smooth muscle relaxation. There is limited literature about their effect on the mechanical performance of the aortic wall. Therefore, the authors determined the influence of hyperthyroidism on the mechanical properties and histomorphological structure of the descending thoracic aorta in rats. Severe hyperthyroidism was induced in 20 male Wistar rats by administering L-thyroxine (T(4)) in their drinking water for 8 weeks; age-matched normal euthyroid rats acted as controls. Animals were sacrificed, and the mechanical and histomorphometrical characteristics of the descending thoracic aorta were studied. The aortic wall of hyperthyroid rats was stiffer than that of euthyroid animals at the upper physiologic levels of stress or strain (p < 0.05) but less stiff at the lower physiologic and lower levels (p < 0.05). The aorta of hyperthyroid animals compared with that of euthyroid ones showed an increase of the internal and external diameters (p < 0.05), the media area (p < 0.05), the number of smooth muscle cell nuclei (p < 0.05), and the collagen density (p < 0.05) and a decrease in the elastin laminae thickness (p < 0.001) and elastin density (p < 0.001). In hyperthyroid rats, the aortic wall was stiffer at the upper physiologic and higher levels of stress and strain. These changes correlated with microstructural changes of the aortic wall. The coexistence of hyperthyroidism with disease states or clinical conditions that predispose to increased arterial pressure may be associated with increased arterial stiffness and have undesirable consequences on the mechanical performance of the thoracic aorta and hemodynamic homeostasis. These changes could lead to an increased risk for developing vascular complications.