Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries.

Atherosclerosis is rare in internal thoracic arteries (ITA) even in patients with severe atherosclerotic coronary artery (ACA) disease. To explore cellular differences, ITA SMC from 3 distinct donors and ACA SMC from 3 distinct donors were grown to sub-confluence and growth arrested for 48 h. Proliferation and thrombospondin-1 (TSP1) production were determined using standard techniques. ITA SMC were larger, grew more slowly and survived more passages than ACA SMC. ACA SMC had a more pronounced proliferative response to 10% serum than ITA SMC. Both ACA SMC and ITA SMC proliferated in response to exogenous TSP1 (12.5 µg/ml and 25 µg/ml) and platelet derived growth factor-BB (PDGF-BB; 20 ng/ml) but TSP1- and PDGF-BB-induced proliferation were partially inhibited by anti-TSP1 antibody A4.1, microRNA-21(miR-21)-3p inhibitors and miR-21-5p inhibitors in each of the 3 ACA SMC lines, but not in any of the ITA SMC lines. PDGF-BB stimulated TSP1 production in ACA SMC but not in ITA SMC but there was no increase in TSP1 levels in conditioned media in either SMC type. In summary, there are significant differences in morphology, proliferative capacity and in responses to TSP1 and PDGF-BB in SMC derived from ITA compared to SMC derived from ACA.

Metformin and empagliflozin modulate monoamine oxidase-related oxidative stress and improve vascular function in human mammary arteries.

Monoamine oxidases (MAOs), mitochondrial enzymes with two isoforms, A and B, have been recently recognized as significant contributors to oxidative stress in the cardiovascular system. The present study was purported to assess the effect of metformin and empagliflozin on MAO expression, oxidative stress and vascular reactivity in internal mammary arteries harvested from overweight patients with coronary heart disease subjected to bypass grafting. Vascular rings were prepared and acutely incubated (12 h) with high glucose (GLUC, 400 mg/dL) or angiotensin II (AII, 100 nM) and metformin (10 µM) and/or empagliflozin (10 µM) and used for the assessment of MAO expression (qRT-PCR and immune histochemistry), reactive oxygen species (ROS, confocal microscopy and spectrophotometry), and vasomotor function (myograph). Ex vivo stimulation with GLUC or AII increased both MAOs expression, ROS production and impaired relaxation to acetylcholine (ACh) of the vascular rings. All effects were alleviated by incubation with each antidiabetic drug; no cumulative effect was obtained when the drugs were applied together. In conclusion, MAO-A and B are upregulated in mammary arteries after acute stimulation with GLUC and AII. Endothelial dysfunction and oxidative stress were alleviated by either metformin or empagliflozin in both stimulated and non-stimulated vascular samples harvested from overweight cardiac patients.

The Functional Effects of Visfatin on Human Left Internal Mammary Artery.

ABSTRACT: Visfatin may play a role in vascular dysfunction in metabolic disorders. Apart from its insulin-mimetic actions, it has divergent actions in the cardiovascular system with discordant results in the literature. Thus, we aimed to study the effects of visfatin on vascular responses of the human left internal mammary artery. Sections of redundant human left internal mammary artery were cut into 3-mm wide rings and hung in 20-mL organ baths containing physiologic salt solution and attached to an isometric force transducer connected to a computer-based data acquisition system. Removing endothelium caused an increase in pD2 values for visfatin-induced relaxation responses (10 -12 -10 -7 M) (9.06 ± 0.21 and 11.08 ± 0.92, respectively). Nicotinamide phosphoribosyltransferase inhibitor FK866 (10 µM) reversed the visfatin-induced relaxations (10 -12 -10 -7 M) ( P = 0.024). Incubations with nitric oxide synthase inhibitor nitro- l -arginine methylester and guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) caused significant reductions in relaxation responses of visfatin ( P = 0.011 and 0.008, respectively). Visfatin incubations decreased relaxation responses to acetylcholine but not to sodium nitroprusside. Incubations with visfatin did not change contractile responses to angiotensin II, endothelin-1, noradrenalin, and phenylephrine. In this study, visfatin caused endothelium-dependent relaxations mediated by nitric oxide-cyclic guanosine monophosphate pathway and nicotinamide phosphoribosyltransferase activity. Furthermore, visfatin-induced decreases in relaxation responses were also related to endothelium-derived nitric oxide.

Comparative study of coronary artery bypass graft materials: reduced contraction and ADMA levels in internal mammary artery versus saphenous vein.

BACKGROUND: Vasospasm and atherosclerosis due to low endothelial capacity are the most important causes of coronary artery bypass graft failure observed in internal mammary artery (IMA) and saphenous vein (SV). Vasospasm can be mimicked in in-vitro studies by inducing vasoconstriction of graft materials. In the present study, we aimed to compare the vascular contraction induced by several spasmogens including prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2α), phenylephrine (PE), leukotriene C4 (LTC4), LTD4, potassium chloride (KCl), and arachidonic acid between IMA and SV preparations. Furthermore, endothelial capacity, nitrite and asymmetric dimethylarginine (ADMA) levels were compared between two grafts. METHODS: By using organ bath, contractile responses induced by different spasmogens were compared between IMA and SV preparations derived from patients underwent coronary artery bypass surgery (N.=35). The endothelial capacity was determined by acetylcholine-induced (ACh) relaxation in PE-precontracted vessels. Nitrite and ADMA levels were measured in organ culture supernatant of IMA and SV preparations. RESULTS: Contractile responses induced by PGE2, PGF2α, PE, LTC4, LTD4, KCl and arachidonic acid were significantly lower in IMA preparations versus SV preparations. ACh-induced relaxation was significantly more prominent in IMA than SV preparations. Nitrite levels were greater and ADMA levels were lower in IMA versus SV preparations. CONCLUSIONS: IMA has reduced capacity to constrict to several vasoconstrictor agents. Furthermore, IMA has greater endothelial capacity associated with higher nitrite levels and lower ADMA levels. Our results support the greater patency rate observed in IMA versus SV preparations.

Comparative study on the effect of aspirin, TP receptor antagonist and TxA2 synthase inhibitor on the vascular tone of human saphenous vein and internal mammary artery.

AIMS: Thromboxane (TxA2) is synthesized from arachidonic acid (AA) via thromboxane synthase (TxS) enzyme and induces vasoconstriction via TP receptor. Our aim is to compare the effects of aspirin, TxS inhibitor and TP receptor antagonist on vascular reactivity of bypass grafts (saphenous vein and internal mammary artery). MAIN METHODS: Using isolated organ bath, saphenous vein and internal mammary artery preparations were incubated with TP receptor antagonist, TxS inhibitor, aspirin, IP or EP4 receptor antagonist. Then prostaglandin (PG)E2, PGF2α, phenylephrine and AA were administered in concentration-dependent manner. The expression of prostanoid receptor and PGI2 synthase (PGIS) enzyme was determined by Western Blot. KEY FINDINGS: TP receptor antagonist inhibited the contraction induced by PGE2, PGF2α, and AA but not that induced by phenylephrine in both types of vessels. Aspirin increased phenylephrine-induced contraction only in internal mammary artery and decreased AA-induced contraction in saphenous vein. TxS inhibitor decreased both PGE2 and AA-induced contraction in both types of vessels. This decrease was reversed by co-incubation of TxS inhibitor and IP/EP4 receptor antagonists. The expressions of EP3 receptor and PGIS enzyme were greater in internal mammary artery compared to saphenous vein while IP and TP receptors expressed at similar levels. SIGNIFICANCE: TP receptor antagonist and TxS inhibitor are more effective to reduce contraction induced by different spasmogens in comparison to aspirin. Our results suggest that TP receptor antagonist and TxS inhibitor might have an advantage over aspirin due to their preventive effect on increased vascular reactivity observed in post-operative period of coronary artery bypass grafting.

Resilience of the Internal Mammary Artery to Atherogenesis: Shifting From Risk to Resistance to Address Unmet Needs.

Fueled by the global surge in aging, atherosclerotic cardiovascular disease reached pandemic dimensions putting affected individuals at enhanced risk of myocardial infarction, stroke, and premature death. Atherosclerosis is a systemic disease driven by a wide spectrum of factors, including cholesterol, pressure, and disturbed flow. Although all arterial beds encounter a similar atherogenic milieu, the development of atheromatous lesions occurs discontinuously across the vascular system. Indeed, the internal mammary artery possesses unique biological properties that confer protection to intimal growth and atherosclerotic plaque formation, thus making it a conduit of choice for coronary artery bypass grafting. Its endothelium abundantly expresses nitric oxide synthase and shows accentuated nitric oxide release, while its vascular smooth muscle cells exhibit reduced tissue factor expression, high tPA (tissue-type plasminogen activator) production and blunted migration and proliferation, which may collectively mitigate intimal thickening and ultimately the evolution of atheromatous plaques. We aim here to provide insights into the anatomy, physiology, cellular, and molecular aspects of the internal mammary artery thereby elucidating its remarkable resistance to atherogenesis. We propose a change in perspective from risk to resilience to decipher mechanisms of atheroresistance and eventually identification of novel therapeutic targets presently not addressed by currently available remedies.

Calcium-activated potassium channel family in coronary artery bypass grafts.

OBJECTIVES: We examined the expression, distribution, and contribution to vasodilatation of the calcium-activated potassium (KCa) channel family in the commonly used coronary artery bypass graft internal thoracic artery (ITA) and saphenous vein (SV) to understand the role of large conductance KCa (BKCa), intermediate-conductance KCa (IKCa), and small-conductance KCa (SKCa) channel subtypes in graft dilating properties determined by endothelium-smooth muscle interaction that is essential to the postoperative performance of the graft. METHODS: Real-time polymerase chain reaction and western blot were employed to detect the messenger RNA and protein level of KCa channel subtypes. Distribution of KCa channel subtypes was examined by immunohistochemistry. KCa subtype-mediated vasorelaxation was studied using wire myography. RESULTS: Both ITA and SV express all KCa channel subtypes with each subtype distributed in both endothelium and smooth muscle. ITA and SV do not differ in the overall expression level of each KCa channel subtype, corresponding to comparable relaxant responses to respective subtype activators. In ITA, BKCa is more abundantly expressed in smooth muscle than in endothelium, whereas SKCa exhibits more abundance in the endothelium. In comparison, SV shows even distribution of KCa channel subtypes in the 2 layers. The BKCa subtype in the KCa family plays a significant role in vasodilatation of ITA, whereas its contribution in SV is quite limited. CONCLUSIONS: KCa family is abundantly expressed in ITA and SV. There are differences between these 2 grafts in the abundance of KCa channel subtypes in the endothelium and the smooth muscle. The significance of the BKCa subtype in vasodilatation of ITA may suggest the potential of development of BKCa modulators for the prevention and treatment of ITA spasm during/after coronary artery bypass graft surgery.

Co-Culture of Primary Human Coronary Artery and Internal Thoracic Artery Endothelial Cells Results in Mutually Beneficial Paracrine Interactions.

Although saphenous veins (SVs) are commonly used as conduits for coronary artery bypass grafting (CABG), internal thoracic artery (ITA) grafts have significantly higher long-term patency. As SVs and ITA endothelial cells (ECs) have a considerable level of heterogeneity, we suggested that synergistic paracrine interactions between CA and ITA ECs (HCAECs and HITAECs, respectively) may explain the increased resistance of ITA grafts and adjacent CAs to atherosclerosis and restenosis. In this study, we measured the gene and protein expression of the molecules responsible for endothelial homeostasis, pro-inflammatory response, and endothelial-to-mesenchymal transition in HCAECs co-cultured with either HITAECs or SV ECs (HSaVECs) for an ascending duration. Upon the co-culture, HCAECs and HITAECs showed augmented expression of endothelial nitric oxide synthase (eNOS) and reduced expression of endothelial-to-mesenchymal transition transcription factors Snail and Slug when compared to the HCAEC-HSaVEC model. HCAECs co-cultured with HITAECs demonstrated an upregulation of HES1, a master regulator of arterial specification, of which the expression was also exclusively induced in HSaVECs co-cultured with HCAECs, suggestive of their arterialisation. In addition, co-culture of HCAECs and HITAECs promoted the release of pro-angiogenic molecules. To conclude, co-culture of HCAECs and HITAECs results in reciprocal and beneficial paracrine interactions that might contribute to the better performance of ITA grafts upon CABG.

Vascular activity of infusion and fractions of Cymbopogon citratus (DC) Stapf. in human arteries.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon citratus (DC.) Stapf has been traditionally used mainly for inflammatory diseases and hypertension. However, the mechanisms underlying its vascular activity remain to be fully characterized and the fractions responsible for its cardiovascular activity are still unknown. AIM OF THE STUDY: In this study, we aimed to assess the vascular activity of Cymbopogon citratus in human arteries and to study the role of cyclooxygenase in its vasorelaxant effects. MATERIALS AND METHODS: Vascular effects of leaves infusion and three fractions (phenolic acids, flavonoids and tannins) were studied using distal segments of human internal thoracic arteries harvested from patients undergoing coronary revascularization, which were mounted as rings in tissue organ baths and maintained at 37 °C in Krebs Henseleit buffer. The effect on basal vascular tone, the effect on the noradrenaline-induced contraction and the vasorelaxant effects were assessed. The role of cyclooxygenase was evaluated with indomethacin. RESULTS: Our results showed a mild effect on the basal vessel tone of the infusion. A significant inhibition on the adrenergic-mediated vasoconstriction was observed for the infusion (0.0002 mg/mL) and the flavonoid fraction (0.2 mg/mL), despite a potentiation was observed in some conditions. A vasorelaxant effect was observed for both the infusion (6.46% of maximal relaxation) and the tannin fraction (26.91% of maximal relaxation, P < 0.05 vs. infusion). Incubation with indomethacin (10 µM) elicited a decrease in the vasorelaxation to the infusion (P < 0.05). CONCLUSIONS: These results suggest that cyclooxygenase may be involved in the vasorelaxation to the infusion of Cymbopogon citratus and that tannins are the compound fraction mainly responsible for this vasorelaxation.

Preventing treatment failures in coronary artery disease: what can we learn from the biology of in-stent restenosis, vein graft failure, and internal thoracic arteries?

Coronary artery disease (CAD) remains one of the most important causes of morbidity and mortality worldwide, and the availability of percutaneous or surgical revascularization procedures significantly improves survival. However, both strategies are daunted by complications which limit long-term effectiveness. In-stent restenosis (ISR) is a major drawback for intracoronary stenting, while graft failure is the limiting factor for coronary artery bypass graft surgery (CABG), especially using veins. Conversely, internal thoracic artery (ITA) is known to maintain long-term patency in CABG. Understanding the biology and pathophysiology of ISR and vein graft failure (VGF) and mechanisms behind ITA resistance to failure is crucial to combat these complications in CAD treatment. This review intends to provide an overview of the biological mechanisms underlying stent and VGF and of the potential therapeutic strategy to prevent these complications. Interestingly, despite being different modalities of revascularization, mechanisms of failure of stent and saphenous vein grafts are very similar from the biological standpoint.

Sex differences in vascular reactivity of coronary artery bypass graft conduits.

Females have increase in-hospital mortality and poorer outcomes following coronary artery bypass grafting (CABG). Biological differences in the reactivity of the graft conduits to circulating catecholamine may contribute to this sex difference. This study examined sex differences in the vasoconstrictor responses of internal mammary artery (IMA) and saphenous vein (SV) conduits to phenylephrine (PE) and endothelin-1 (ET-1). Functional IMA and SV were obtained from 78 male and 50 female patients undergoing CABG (67.7 ± 11 and 69 ± 10 years, respectively) and subjected to the following experimental conditions. (1) Concentration response curves for PE and ET-1 were generated in an intact IMA and SV and endothelium denuded IMA segments, (2) in the presence of the nitric oxide synthase inhibitor (L-NAME) or the cyclooxygenase inhibitor (indomethacin) in an endothelium-intact IMA and (3) the activity state (abundance and phosphorylation) of the α1-adrenergic receptor was investigated using Phos-tag™ western blot analysis. (1) Compared to male, female IMA and SV were hypersensitive to PE but not ET-1 (p < 0.05). The female IMA hypersensitivity response to PE was abolished following endothelial denudation, (2) persisted in the presence of L-NAME but was abolished in the presence of indomethacin and (3) there was no sex differences in the abundance and phosphorylation of the α1-adrenergic receptor in IMA. Female IMA and SV graft conduits are hypersensitive to α1-adrenergic stimuli. This endothelial cyclooxygenase pathway-mediated hypersensitivity may produce excessive IMA and SV graft constriction in females administered catecholamines and could contribute to their poorer CABG outcomes.

Estrogen determines sex differences in adrenergic vessel tone by regulation of endothelial ß-adrenoceptor expression.

Vessels of female rats constrict less and relax more to adrenergic stimulation than vessels of males. Although we have reported that these sex-specific differences rely on endothelial ß-adrenoceptors, the role of sex hormones in ß-adrenoceptor expression and related vessel tone regulation is unknown. We investigated the role of estrogen, progesterone and testosterone on ß-adrenoceptor expression and adrenergic vessel tone regulation, along with sex-specific differences in human mammary arteries. The sex-specific differences in vasoconstriction and vasorelaxation in rat vessels were eliminated after ovariectomy in females. Ovariectomy increased vessel vasoconstriction to norepinephrine more than twofold. Vasorelaxations by isoprenaline and a ß3-agonist were reduced after ovariectomy. Estrogen, but not progesterone substitution, restored sex-specific differences in vasoconstriction and vasorelaxation. Vascular mRNA levels of ß1- and ß3- but not ß2-adrenoreceptors were higher in vessels of females compared with males. Ovariectomy reduced these differences by decreasing ß1- and ß3- but not ß2-adrenoreceptor expression in females. Consistently, estrogen substitution restored ß1- and ß3-adrenoreceptor expression. Orchiectomy or testosterone treatment affected neither vasoconstriction and vasorelaxation nor ß-adrenoceptor expression in vessels of male rats. In human mammary arteries, sex-specific differences in vasoconstriction and vasorelaxation were reduced after removal of endothelium or treatment with l-NMMA. Vessels of women showed higher levels of ß1- and ß3-adrenoceptors than in men. In conclusion, the sex-specific differences in vasoconstriction and vasorelaxation are common for rat and human vessels. In rats, these differences are estrogen but not testosterone or progesterone dependent. Estrogen determines these differences via regulation of vascular endothelial ß1- and ß3-adrenoreceptor expression. NEW & NOTEWORTHY This study proposes a mechanistic concept regulating sex-specific differences in adrenergic vasoconstriction and vasorelaxation. Estrogen increases vascular ß1- and ß3-adrenoceptor expression in female rats. This and our previous studies demonstrate that these receptors are located primarily on endothelium and when activated by norepinephrine act via nitric oxide (NO). Therefore, ß-adrenergic stimulation leads to a more pronounced vasorelaxation in females. Coactivation of endothelial ß1- and ß3-adrenoreceptors leads to higher NO release in vessels of females, ultimately blunting vasoconstriction triggered by activation of smooth muscle α-adrenoceptors.

RhoA/rho-kinase, nitric oxide and inflammatory response in LIMA during OPCABG with isoflurane preconditioning.

BACKGROUND: Grafting vessel with LIMA to the left anterior descending coronary artery plays a most important role in the long-term prognosis of OPCABG surgery. The aim of this study was to compare the effects of isoflurane preconditioning on miRs and mRNAs levels in the left internal mammary arterie (LIMA) graft with propofol in patients undergoing off-pump coronary artery bypass surgery (OPCABG). METHODS: Patients were randomly assigned to receive either propofol (n = 15), or interrupted isoflurane (n = 15). In group P, propofol administration was continued at 3-5 mg/kg/h intravenous injection for the duration of surgery. Five minutes prior to incision, patients of the isoflurane group (group Iso) received 2 cycles of 1 MAC isoflurane. RESULTS: miR-221 were significantly lower in group Iso (P < 0 .05). E-selectin mRNA, RhoA mRNA and ROK mRNA were significantly lower at specimens of LIMA in group Iso compared with those in group P patients (P < 0 .05). The expression of NOS3 mRNA was significantly higher in group Iso patients (P < 0 .05). CONCLUSION: Our findings provide some insight that prior interrupted isoflurane administration could regulate miR-221, and downstream effectors (mRNAs) and resulted in actual attenuation of inflammation and spasm of LIMA in patients undergoing OPCABG surgery. TRIAL REGISTRATION: NCT No. ( ClinicalTrials.gov ): NCT02678650; Registration date: January 23, 2016.

Inflammation increases MMP levels via PGE2 in human vascular wall and plasma of obese women.

BACKGROUND AND OBJECTIVES: Matrix metalloproteinases (MMPs) are involved in several inflammatory processes including obesity-related vascular diseases and graft failure of coronary artery (CA) bypass grafts [internal mammary artery (IMA), saphenous vein (SV)]. In these inflammatory conditions, the release of prostaglandin E2 (PGE2) is increased via the activity of inducible microsomal PGE synthase-1 (mPGES-1). Our aim was to investigate whether MMPs and their endogenous inhibitor (TIMPs) may be regulated by PGE2 under inflammatory conditions in human vasculature and perivascular adipose tissue (PVAT), as well as in plasma of obese patients. METHODS: MMP-1,-2 and TIMP-1,-2 densities were measured in human plasma (n = 68) as well as in supernatants of human vascular wall (IMA n = 16, SV n = 14, CA n = 13) and their PVAT. The effects of inflammation and mPGES-1 inhibitor (Compound III, 10 µM) on MMPs regulation were evaluated. The correlations between PGE2 and several parameters were calculated in plasma from patients with or without obesity. RESULTS: The vascular wall and PVAT from SV exhibited the greatest MMP-1,-2 release. An increase of MMP-1,-2 and/or a decrease of TIMP-1 quantities have been detected under inflammation only in vascular wall not in PVAT. These changes under inflammation were completely reversed by inhibition of mPGES-1. In obesity, C-reactive protein (CRP), biomarker of inflammation, and PGE2 levels were increased. PGE2 contents were positively correlated with some anthropometric parameters and plasmatic CRP in both genders, while the correlation with the plasmatic MMP-1 density was significant only in women. CONCLUSIONS: The greater MMP activity observed in SV may contribute to the increased prevalence of graft failure. Under inflammation, the greater mPGES-1 and PGE2 levels lead to enhanced MMP activity in human vascular walls. The positive association between PGE2 and MMP-1 or CRP has been observed in plasma of women. We suggest that mPGES-1 inhibitors could prevent graft failure and obesity-related vascular remodeling mostly in women.

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.

Is internal thoracic artery resistant to reperfusion injury? Evaluation of the storage of free internal thoracic artery grafts.

OBJECTIVES: The in situ internal thoracic artery (ITA) is recognized as the best conduit for coronary artery bypass surgery. The ITA-if it is used as an in situ graft-has a much higher late patency rate than any other arterial graft, including a free ITA graft. We sought to determine if the use of the ITA as an in situ/free graft and its storage in preservation solutions, have an effect on endothelial function. METHODS: The ITA was harvested as either a free or in situ graft in a porcine model. Free grafts were stored in different preservation solutions (saline, Custodiol and Tiprotec [both Köhler Chemie GmbH, Bensheim, Germany]). The ITA was anastomosed off pump to the left anterior descending artery (as in situ/free graft). Freshly harvested ITA served as a control. After 2 hours of reperfusion, the implanted grafts were harvested. The assessment of endothelial function, histopathological analysis, and gene expression were performed. RESULTS: Endothelial function and integrity were severely impaired after reperfusion in the free ITA groups, however, it was partially preserved in the Tiprotec group. Reperfusion injury resulted in increased nitro-oxidative stress, DNA breakage, vascular cell adhesion protein 1, intercellular adhesion molecule-1, and caspase-3 scores, and a decreased endothelial nitric oxide synthase score in the free ITA groups. The in situ ITA graft showed no signs of injury. mRNA levels were significantly altered among the groups. CONCLUSIONS: An early, severe endothelial dysfunction of the stored, free ITA as described, could be completely prevented by the use of an in situ ITA graft. Tiprotec might be a feasible option for storage of free arterial grafts during coronary artery bypass grafting.

Perfusion Tissue Culture Initiates Differential Remodeling of Internal Thoracic Arteries, Radial Arteries, and Saphenous Veins.

Adaptive remodeling processes are essential to the maintenance and viability of coronary artery bypass grafts where clinical outcomes depend strongly on the tissue source. In this investigation, we utilized an ex vivo perfusion bioreactor to culture porcine analogs of common human bypass grafts: the internal thoracic artery (ITA), the radial artery (RA), and the great saphenous vein (GSV), and then evaluated samples acutely (6 h) and chronically (7 days) under in situ or coronary-like perfusion conditions. Although morphologically similar, primary cells harvested from the ITA illustrated lower intimal and medial, but not adventitial, cell proliferation rates than those from the RA or GSV. Basal gene expression levels were similar in all vessels, with only COL3A1, SERPINE1, FN1, and TGFB1 being differentially expressed prior to culture; however, over half of all genes were affected nominally by the culturing process. When exposed to coronary-like conditions, RAs and GSVs experienced pathological remodeling not present in ITAs or when vessels were studied in situ. Many of the remodeling genes perturbed at 6 h were restored after 7 days (COL3A1, FN1, MMP2, and TIMP1) while others (SERPINE1, TGFB1, and VCAM1) were not. The findings elucidate the potential mechanisms of graft failure and highlight strategies to encourage healthy ex vivo pregraft conditioning.

Sphingosine-1-phosphate receptor 1 regulates neointimal growth in a humanized model for restenosis.

OBJECTIVE: The main objective of this study was to define a role of sphingosine-1-phosphate receptor 1 (S1PR1) in the arterial injury response of a human artery. The hypotheses were tested that injury induces an expansion of S1PR1-positive cells and that these cells accumulate toward the lumen because they follow the sphingosine-1-phosphate gradient from arterial wall tissue (low) to plasma (high). METHODS: A humanized rat model was used in which denuded human internal mammary artery (IMA) was implanted into the position of the abdominal aorta of immunosuppressed Rowett nude rats. This injury model is characterized by medial as well as intimal hyperplasia, whereby intimal cells are of human origin. At 7, 14, and 28 days after implantation, grafts were harvested and processed for fluorescent immunostaining for S1PR1 and smooth muscle α-actin. Nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride. Using digitally reconstructed, complete cross sections of grafts, intimal and medial areas were measured, whereby the medial area had virtually been divided into an outer (toward adventitia) and inner (toward lumen) layer. The fraction of S1PR1-positive cells was determined in each layer by counting S1PR1-positive and S1PR1-negative cells. RESULTS: The fraction of S1PR1-postive cells in naive IMA is 58.9% ± 6.0% (mean ± standard deviation). At day 28 after implantation, 81.6% ± 4.4% of medial cells were scored S1PR1 positive (P < .01). At day 14, the ratio between S1PR1-positive and S1PR1-negative cells was significantly higher in the lumen-oriented inner layer (9.3 ± 2.1 vs 6.0 ± 1.0; P < .01). Cells appearing in the intima at day 7 and day 14 were almost all S1PR1 positive. At day 28, however, about one-third of intimal cells were scored S1PR1 negative. CONCLUSIONS: From these data, we conclude that denudation of IMA specifically induces the expansion of S1PR1-positive cells. Based on the nonrandom distribution of S1PR1-positive cells, we consider the possibility that much like lymphocytes, S1PR1-positive smooth muscle cells also use S1PR1 to recognize the sphingosine-1-phosphate gradient from tissue (low) to plasma (high) and so migrate out of the media toward the intima of the injured IMA.

No detectable differential microRNA expression between non-atherosclerotic arteries of type 2 diabetic patients (treated or untreated with metformin) and non-diabetic patients.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is an independent risk factor of cardiovascular disease (CVD), however, the underlying mechanisms are largely unknown. Using non-atherosclerotic internal thoracic arteries (ITAs) obtained from coronary artery bypass grafting, we previously identified a distinct elevation in the level of proteins comprising the arterial basement membrane in T2DM patients not treated with metformin. Altered transcription of genes encoding these proteins has not been observed, indicating alternative mechanisms of dysregulation. METHODS: In this study we screened for differential expression of arterial microRNAs (miRNAs) in T2DM patients to test the hypothesis that the arterial protein signature of diabetic patients is associated with dysregulation at the miRNA level, and further to lay the foundation for novel hypotheses addressing the increased CVD risk of T2DM patients. MiRNA isolated from fresh frozen ITAs [from 18 T2DM- (10 of which were subject to metformin treatment) and 30 non-diabetes mellitus (non-DM) patients] were analyzed by microarray, and miRNAs isolated from formalin-fixated paraffin-embedded (FFPE) ITAs were analyzed by quantitative PCR (qPCR) in an independent study group [26 T2DM- (15 of which were subject to metformin treatment) and 26 non-DM patients] to determine expression levels of miRNAs in a pre-defined panel of 12 miRNAs. RESULTS: Unexpectedly, no miRNAs were found to be affected by T2DM status in either of the two study groups. CONCLUSIONS: Our data suggest that alternatives to microRNA dysregulation underlie T2DM-associated protein changes in non-atherosclerotic arteries.