Investigation of red blood cell and platelet indices in adult dogs suffered from myxomatous mitral valve disease with and without pulmonary hypertension.

Background: Pulmonary hypertension (PH) is a common complication of cardiopulmonary disease. In dogs, PH commonly occurs secondary to myxomatous mitral valve disease (MMVD). Red blood cell and platelet indices including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), mean platelet volume (MPV) and platelet distribution width (PDW), have previously been found to be indicators for predicting and prognosing PH in humans. Therefore, this study aimed to investigate whether these indices are associated with MMVD and/or PH in dogs. Methods: Two hundred and forty-six dogs were retrospectively recruited for the study and classified into 4 groups: normal (n = 49), MMVD (n =102), PH (n =17), MMVD+PH (n =78). A sub-analysis was performed in dogs with MMVD without evidence of PH according to stage B1 (n =20), stage B2 (n =15), stage C (n =67). The data are expressed as median (interquartile range). Results and discussion: No significant differences (p < 0.05) were found in MCV, RDW and MPV among all groups (normal, MMVD, PH and MMVD+PH). However, decreases in MCH and MCHC were found in MMVD [22.40 (20.90-23.50) pg and 35.25 (33.08-36.90) g/dL], MMVD+PH [22.25 (20.85-23.98) pg and 35.65 (33.30-37.33) g/dL] and PH groups [21.20 (20.60-22.20) pg and 33.80 (32.75-35.70) g/dL] compared to the normal dogs [24.29 (23.55-24.90) pg and 38.20 (37.50-39.05) g/dL] (p < 0.001). Decreases in PDW were found in dogs in the MMVD+PH [15.10 (14.98-15.30) %] groups compared to dogs in the normal group [15.30 (15.10-15.50) %] (p = 0.004). Sub-analysis of MMVD dogs without PH showed a decrease in MCH in dogs with stage B2 MMVD [21.00 (20.50-22.90) pg] and stage C MMVD [22.40 (20.90-23.20) pg] compared to normal dogs [24.29 (23.55-24.90) pg] (p < 0.001). MCHC of dogs with stage B1 [36.55 (33.53-37.78) g/dL] (p = 0.004), B2 [32.90 (32.00-35.00) g/dL] (p < 0.001) and C MMVD [35.30 (33.30-36.80) g/dL] (p < 0.001) were lower than those of normal dogs [38.20 (37.50-39.05) g/dL]. PDW in the stage C MMVD group [15.10 (15.00-15.30) %] was reduced compared to the normal group [15.30 (15.10-15.50) %] (p = 0.042) and the stage B1 MMVD group [15.35 (15.23-15.68) %] (p = 0.002). MCH, MCHC and PDW were negatively correlated with the left atrial and left ventricular size. Conclusion: Decreases in MCH and MCHC are related to MMVD, precapillary PH and postcapillary PH while PDW are associated with MMVD severity but not with the presence of PH.

Assessment of microcirculation variables and endothelial glycocalyx using sidestream dark field videomicroscopy in anesthetized dogs undergoing cardiopulmonary bypass.

Introduction: To evaluate microcirculation and endothelial glycocalyx (eGC) variables using sidestream darkfield (SDF) videomicroscopy in canine cardiopulmonary bypass (CPB). Methods: Dogs undergoing CPB for surgical correction of naturally-occurring cardiac disease were prospectively included. Variables collected included patient demographics, underlying cardiac disease, red blood cell flow (Flow), 4-25 µm vessel density (Density), absolute capillary blood volume (CBVabs), relative capillary blood volume (CBVrel) and eGC width assessed by perfused boundary region (PBR). Anesthetized healthy dogs were used as control. Microcirculation and eGC variables were compared at baseline under anesthesia (T0), on CPB prior to cross clamping (T1), after cross clamp removal following surgical correction (T2) and at surgical closure (T3). Results: Twelve dogs were enrolled, including 10 with a complete dataset. Median Flow was 233.9, 79.9, 164.3, and 136.1 µm/s at T0, T1, T2, and T3, respectively, (p = 1.00). Median Density was 173.3, 118.4, 121.0 and 155.4 mm/mm2 at T0, T1, T2, and T3, respectively, (p = 1.00). Median CBVabs decreased over time: 7.4, 6.6, 4.8 and 4.7 103µm3 at T0, T1, T2, and T3, respectively, (p < 0.01). Median CBVrel increased over time: 1.1, 1.5,1.1, and 1.3 103µm3 at T0, T1, T2, and T3, respectively, (p < 0.001). Median PBR increased over time: 1.8, 2.1, 2.4, 2.1 µm at T0, T1, T2, and T3, respectively, (p < 0.001). Compared to control dogs (n = 8), CPB dogs had lower CBVabs at T0. Conclusion: Alterations in eGC thickness and microvascular occur in dogs undergoing CPB for naturally-occurring cardiac disease.

Surgical repair for canine tricuspid valve dysplasia: Technique and case report.

Tricuspid valve dysplasia (TVD) is a congenital malformation of the right atrioventricular valve characterized by restricted leaflet motion, annular dilation, and tricuspid regurgitation (TR). Severe cases typically exhibit progressive right-sided congestive heart failure, affecting the quality of life and survival. This article describes a technique for surgical repair of TVD and a case report with long-term follow-up. A 1.5-year-old intact male Labrador retriever with severe TR underwent surgical repair for TVD. Valve repair was performed under cardiopulmonary bypass and consisted of neochord mobilization of the valve leaflets and partial band annuloplasty. Transthoracic echocardiogram performed 5 days after surgery showed mild TR, a 93% decrease in anatomic regurgitant orifice area, and decreased right chamber dimensions. Forty-eight months after repair, the patient was free of clinical signs, did not have a heart murmur, and was receiving no cardiac medications. Based on this case, surgical repair of TVD is feasible with long-term durability, and the outcome suggests that the described technique may be a viable treatment option for patients with severe TVD.

Epicardial pacemaker implantation in small animals.

Epicardial pacemaker implantation can be performed as a lone procedure or in combination with another thoracic or abdominal surgery. This article reviews the current literature and describes a minimally invasive approach for epicardial pacemaker implantation in small animals. The principal advantage of epicardial pacing is that it avoids contact with blood and intracardiac structures and thereby avoids uncommon but potentially devastating complications associated with endocardial pacemaker implantation. Epicardial pacing as a lone procedure can be accomplished via an abdominal transdiaphragmatic or minimal incision thoracotomy approach (minithoracotomy). A minithoracotomy offers the advantages of being less invasive and providing more direct access to the cardiac surface for suturing of epicardial electrodes. Epicardial pacing is a viable option for smaller animals, animals with pre-existing infections, animals at risk for thrombotic complications, or animals undergoing another thoracic or abdominal surgery.

Inflow Venous Occlusion for Intracardiac Resection of an Occluding Right Ventricular Tumor.

Use of normothermic venous inflow occlusion enabled removal of an intracardiac tumor in a 4 yr old, 27 kg, spayed female Airedale terrier with a history of appendicular osteosarcoma and recent exertional syncope. Inflow venous occlusion via a median sternotomy thoracotomy without hypothermia was used to access the mineralized mass within the right ventricular outflow tract. Duration of circulatory arrest was 70 s for this beating heart surgery. A circumscribed intracardiac chondrosarcoma tumor was marginally resected in this dog, successfully alleviating exertional syncope and restoring a normal echogenic appearance of the right heart. Asymptomatic intracardiac chondrosarcoma recurrence and pulmonary metastasis was detected at 309 days and cardiopulmonary arrest occurred 372 days following intracardiac surgery. Use of inflow occlusion is a viable technique for select intracardiac tumors in dogs with preoperative planning.

Apoptosis and abundance of Bcl-2 family and transforming growth factor ß1 signaling proteins in canine myxomatous mitral valves.

OBJECTIVES: To determine the percentage of cells undergoing apoptosis within canine myxomatous valves and to evaluate whether TGFß1 can be implicated as an anti-apoptosic signal through the Bcl-2 family of signaling proteins. ANIMALS: Post-mortem mitral valve leaflets harvested from 5 normal dogs, 5 dogs with early-stage myxomatous mitral valve disease (MMVD), and 5 dogs with late-stage MMVD. MATERIALS AND METHODS: The number of cells expressing cleaved caspase-3, DNA fragmentation (TUNEL marker) and apoptotic bodies were evaluated as a measure of apoptosis. To evaluate the relationship between TGFß1 signaling and apoptosis, the abundance of activated TGFß1 signaling protein, phosphorylated Smad 2/3 (p-Smad 2/3), and Bcl-2 family proteins (pro-apoptotic Bax and anti-apoptotic Bcl-2) was determined by immunohistochemistry. RESULTS: Cells in normal and both stages of MMVD expressed the TUNEL marker and cleaved caspase-3, but not apoptotic bodies. The percentage of TUNEL marker and cleaved caspase-3 positive nuclei was not significantly different between groups of dogs (p > 0.05). P-Smad 2/3 and Bax were more abundant in myxomatous mitral valves while Bcl-2 was less abundant. P-Smad 2/3 primarily increased in the atrialis layer and was abundantly increased only in late-stage MMVD. CONCLUSIONS: These data suggest that interstitial cells in MMVD are in a pro-apoptotic condition; however, they do not execute apoptosis. Thus, apoptosis does not explain differences in cellular density in canine MMVD. TGFß1 signaling through the canonical SMAD pathway is increased in myxomatous mitral valves, but does not apparently mediate interstitial cell apoptosis in canine MMVD.

Aortic thrombosis in dogs: presentation, therapy, and outcome in 26 cases.

OBJECTIVES: The pathogenesis and presentation of aortic thrombosis (AT) in dogs is not well characterized and an effective antithrombotic therapy for AT in dogs has not been identified. Our goal is to report the clinical presentation and results of therapies in dogs with AT. ANIMALS: Twenty-six client-owned dogs. METHODS: Retrospective review of medical records of dogs diagnosed with AT between 2003 and 2010. RESULTS: Twenty-six dogs had an apparent primary mural aortic thrombus. None had structural heart disease at diagnosis. Twenty dogs were ambulatory with varying degrees of pelvic limb dysfunction. Duration of ambulatory dysfunction was 7.8 weeks (range 1 day-52 weeks). A majority of dogs (58%) had no concurrent conditions at diagnosis. Fourteen dogs were treated with a standard warfarin protocol for a median period of 22.9 months (range 0.5-53 months). Ambulatory function improved in all dogs treated with warfarin. Time until clinical improvement was 13.9 days (range 2-49 days). Dogs treated with warfarin did not become non-ambulatory, die or undergo euthanasia related to AT, or have a known serious hemorrhagic event. CONCLUSIONS: The pathogenesis of AT in dogs is distinct from that of aortic thromboembolism (ATE) in cats. Aortic thrombosis in dogs is more likely to involve local thrombosis in the distal aorta with embolization to the arteries of the pelvic limb resulting in chronic progressive ambulatory dysfunction. Chronic warfarin administration is well-tolerated and appears to be an effective short-term and long-term therapy for dogs with AT.

Signaling pathways in mitral valve degeneration.

Heart valves exhibit a highly-conserved stratified structure exquisitely designed to counter biomechanical forces delivered over a lifetime. Heart valve structure and competence is maintained by heart valve cells through a process of continuous turnover extracellular matrix (ECM). Degenerative (myxomatous) mitral valve disease (DMVD) is an important disease associated with aging in both dogs and humans. DMVD is increasingly regarded as a disease with identifiable signaling mechanisms that control key genes associated with regulation and dysregulation of ECM homeostasis. Initiating stimuli for these signaling pathways have not been fully elucidated but likely include both mechanical and chemical stimuli. Signaling pathways implicated in DMVD include serotonin, transforming growth factor ß (TGFß), and heart valve developmental pathways. High circulating serotonin (carcinoid syndrome) and serotoninergic drugs are known to cause valvulopathy that shares pathologic features with DMVD. Recent evidence supports a local serotonin signaling mechanism, possibly triggered by high tensile loading on heart valves. Serotonin initiates TGFß signaling, which in turn has been strongly implicated in canine DMVD. Recent evidence suggests that degenerative aortic and mitral valve disease may involve pathologic processes that mimic osteogenesis and chondrogenesis, respectively. These processes may be mediated by developmental pathways shared by heart valves, bone, and cartilage. These pathways include bone morphogenic protein (BMP) and Wnt signaling. Other signaling pathways implicated in heart valve disease include Notch, nitric oxide, and angiotensin II. Ultimately, increased understanding of signaling mechanisms could point to therapeutic strategies aimed at slowing or halting disease progression.

Static and cyclic tensile strain induce myxomatous effector proteins and serotonin in canine mitral valves.

OBJECTIVES: Degenerative (myxomatous) mitral valve disease is an important cardiac disease in dogs and humans. The mechanisms that initiate and propagate myxomatous pathology in mitral valves are poorly understood. We investigated the hypothesis that tensile strain initiates expression of proteins that mediate myxomatous pathology. We also explored whether tensile strain could induce the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1), serotonin synthesis, and markers of chondrogenesis. ANIMALS: Mitral valves were obtained postmortem from dogs without apparent cardiovascular disease. METHODS: Mitral valves were placed in culture and subjected to 30% static or cyclic tensile strain and compared to cultured mitral valves subjected to 0% strain for 72 h. Abundance of target effector proteins, TPH1, and chondrogenic marker proteins was determined by immunoblotting. Serotonin was measured in the conditioned media by ELISA. RESULTS: Both static and cyclic strain increased (p < 0.05) expression of myxomatous effector proteins including markers of an activated myofibroblast phenotype, matrix catabolic and synthetic enzymes in canine mitral valves compared to unstrained control. Expression of TPH1 was increased in statically and cyclically strained mitral valves. Expression of chondrogenic markers was increased in statically strained mitral valves. Serotonin levels were higher (p < 0.05) in media of cyclically strained valves compared to unstrained valves after 72 h of culture. CONCLUSION: Static or cyclic tensile strain induces acute increases in the abundance of myxomatous effector proteins, TPH1, and markers of chondrogenesis in canine mitral valves. Canine mitral valves are capable of local serotonin synthesis, which may be influenced by strain.

Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves.

This study addressed the following questions: 1) Does cyclic tensile strain induce protein expression patterns consistent with myxomatous degeneration in mitral valves? 2) Does cyclic strain induce local serotonin synthesis in mitral valves? 3) Are cyclic strain-induced myxomatous protein expression patterns in mitral valves dependent on local serotonin? Cultured sheep mitral valve leaflets were subjected to 0, 10, 20, and 30% cyclic strain for 24 and 72 h. Protein levels of activated myofibroblast phenotype markers, α-smooth muscle actin (α-SMA) and nonmuscle embryonic myosin (SMemb); matrix catabolic enzymes, matrix metalloprotease (MMP) 1 and 13, and cathepsin K; and sulfated glycosaminoglycan (GAG) content in mitral valves increased with increased cyclic strain. Serotonin was present in the serum-free media of cultured mitral valves and concentrations increased with cyclic strain. Expression of the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1) increased in strained mitral valves. Pharmacologic inhibition of the serotonin 2B/2C receptor or TPH1 diminished expression of phenotype markers (α-SMA and SMemb) and matrix catabolic enzyme (MMP1, MMP13, and cathepsin K) expression in 10- and 30%-strained mitral valves. These results provide first evidence that mitral valves synthesize serotonin locally. The results further demonstrate that tensile loading modulates local serotonin synthesis, expression of effector proteins associated with mitral valve degeneration, and GAG synthesis. Inhibition of serotonin diminishes strain-mediated protein expression patterns. These findings implicate serotonin and tensile loading in mitral degeneration, functionally link the pathogeneses of serotoninergic (carcinoid, drug-induced) and degenerative mitral valve disease, and have therapeutic implications.

Bioprosthesis valve replacement in dogs with congenital tricuspid valve dysplasia: technique and outcome.

OBJECTIVE: To describe the surgical technique and report outcome of dogs undergoing bioprosthesis valve replacement for severe tricuspid regurgitation (TR) secondary to congenital tricuspid valve dysplasia (TVD). ANIMALS, MATERIALS AND METHODS: Twelve client-owned dogs (19-43 kg) with TVD underwent tricuspid valve replacement with a bovine pericardial or porcine aortic bioprosthesis with the aid of cardiopulmonary bypass. Anticoagulation with warfarin was maintained for 3 months after surgery and then discontinued. RESULTS: Ten of 12 (83.3%) dogs survived surgery and were discharged from the hospital. Seven dogs were alive with complete resolution of TR for a median period of 48 months (range 1-66 months) after surgery. Two dogs underwent euthanasia because of bioprosthesis failure due to inflammatory pannus at 10 and 13 months after surgery. Two dogs experienced valve thrombosis that was resolved by tissue plasminogen activator. One dog developed suspected endocarditis after surgery that was resolved with antibiotics. Serious cardiac complications included atrial fibrillation and flutter, right-to-left shunt through an uncorrected patent foramen ovale, complete atrioventricular block, and sudden cardiac arrest. Postoperative atrial fibrillation or flutter did not occur in 7 dogs treated prophylactically with oral amiodarone before surgery. CONCLUSIONS: Curative intermediate-term outcomes are possible in dogs undergoing open tricuspid valve replacement with a bioprosthesis. Prosthesis-related complications include inflammatory pannus, thrombosis, and endocarditis. Postoperative atrial fibrillation or flutter can be reduced or prevented by prophylactic preoperative treatment with amiodarone. Several identified complications are avoidable or can be reduced with increased awareness and experience with these techniques.

Serotonin transmembrane transporter is down-regulated in late-stage canine degenerative mitral valve disease.

OBJECTIVE: To compare expression of the serotonin transmembrane transporter (SERT) in normal, early-stage degenerative, and late-stage degenerative canine mitral valve disease. ANIMALS: 24 post-mortem canine mitral valves. METHODS: SERT expression was determined in canine normal (n = 8), early-stage degenerative (n = 8), and late-stage degenerative (n = 8) mitral valves by immunohistochemistry (IHC) and immunoblot (IB) analyses. RESULTS: SERT was expressed in valve interstitial cells of all layers of normal and early-stage degenerative mitral valves based on IHC. SERT was markedly down-regulated in valve interstitial cells, but not valve endothelial cells, of late-stage degenerative mitral valves. SERT expression was significantly decreased in late-stage compared to normal and early-stage degenerative mitral valves based on IB analysis (P < 0.05). CONCLUSIONS: Down-regulation of SERT expression occurs in valve interstitial cells of late-stage, but not early-stage, canine degenerative mitral valves. Down-regulation of SERT could enhance the recently speculated role of serotonin in canine DMVD by decreasing serotonin metabolism and increasing interaction with its receptor. Down-regulation of SERT likely does not play an initiating role in canine DMVD since it does not occur in early-stage disease.

Tryptophan hydroxylase 1 expression is increased in phenotype-altered canine and human degenerative myxomatous mitral valves.

BACKGROUND AND AIM OF THE STUDY: Serotonin is a known mediator of myxomatous pathology in heart valves. Tryptophan hydroxylase 1 (TPH1) is the limiting enzyme for peripheral serotonin synthesis, and its expression by valve interstitial cells (IC) could implicate an autocrine serotonin signaling mechanism in primary degenerative myxomatous mitral valve disease. Thus, the expression of TPH1 in canine and human myxomatous mitral valves was determined, and IC phenotypes expressing TPH1 identified. METHODS: TPH1 expression was determined in canine and human myxomatous and normal mitral valves by immunoblot (IB) and immunofluorescence microscopy (IFM). Co-localization of TPH1 expression with markers of IC phenotype transformation, alpha-smooth muscle actin (a-SMA) and non-muscle embryonic myosin (SMemb) was determined using double-IFM. RESULTS: TPH1 expression by IB was increased (p < 0.05) by three- to five-fold in canine early-stage and late-stage myxomatous valves, and in human surgically excised myxomatous valves compared to canine and human normal control valves, respectively. The number of TPH1 immunopositive cells per x400 field was increased (p < 0.005) in canine (14.9 +/- 1.2) and human (14.9 +/- 2.9) myxomatous valves compared to canine (5.0 +/- 2.4) and human (2.9 +/- 0.6) normal control valves, respectively. Patterns for alpha-SMA and SMemb IC phenotype transformation were distinctly different in myxomatous valves. TPH1 expression was more closely associated with the SMemb IC phenotype in canine and human myxomatous valves. CONCLUSION: An increased expression of TPH1 in canine and human myxomatous mitral valves implicates an autocrine serotonin signaling mechanism in primary degenerative myxomatous mitral valves. TPH1 expression is associated with the SMemb-positive IC phenotype.

Tissue-gel electrophoresis enhances antigen removal from porcine aortic valve and bovine pericardium.

BACKGROUND AND AIM OF THE STUDY: A tissue-engineered heart valve could provide a living prosthesis with characteristics of an ideal valve replacement. One approach to scaffolding a tissue-engineered heart valve is through the 'decellularization' of xenogeneic tissues. Concerns regarding the completeness of antigen removal associated with current detergent-based decellularization treatments have been raised. The study aim was to evaluate antigen removal from candidate xenogeneic bioscaffolds using a novel tissue-gel electrophoresis (TGE) method. METHODS: Porcine aortic valve (PAV) conduit and bovine pericardium (BP) were treated sequentially with hypotonic lysis, sodium dodecyl sulfate (SDS), and TGE. The completeness of antigen removal was evaluated by immunoblot analysis of extractable soluble proteins using rabbit anti-PAV or anti-BP serum. Tissues were also evaluated by hematoxylin and eosin histology. RESULTS: TGE enhanced antigen removal from both the PAV and BP. The effects of TGE were shown to depend on the SDS concentration and voltage (60 versus 120 V), but to be independent of time after 4 h. The effects of TGE were detectable both before and after 96 h aqueous washout. Treatment with 1.0% SDS with TGE (120 V for 4 h) resulted in complete acellularity and no detectable soluble protein antigens from the PAV conduit. CONCLUSION: TGE is a promising adjunctive decellularization method for generating non-immunoreactive bioscaffolds from xenogeneic tissues.

Immunoblot detection of soluble protein antigens from sodium dodecyl sulfate- and sodium deoxycholate-treated candidate bioscaffold tissues.

BACKGROUND AND AIMS OF THE STUDY: The detergent-based 'decellularization' of xenogeneic tissues is one approach to scaffolding a tissue-engineered heart valve construct; however, concern persists regarding the immunogenicity of decellularized xenogeneic bioscaffolds. The study aims were to: (i) develop a sensitive and robust immunoblot-based assay for the detection of soluble protein antigens in xenogeneic bioscaffolds; and (ii) evaluate the completeness of protein antigen removal from sodium dodecyl sulfate (SDS)- or sodium deoxycholate (SD)-treated bovine pericardium (BP) or porcine aortic valve (PAV) conduit. METHODS: Homogenized BP or PAV were injected into rabbits to generate immune serum towards these tissues. Soluble proteins were extracted from untreated BP and PAV. Immunoblot analyses of the extracts were performed using pre-immune and 14-, 28-, 42-, 56- and 70-day post-immune serum. BP and PAV were treated sequentially with 4 h hypotonic lysis; with 0, 0.01, 0.025, 0.05, 0.1, 0.25 or 0.5% SDS or SD for 24 h; and with 96 h of aqueous wash-out. Immunoblot analyses of protein extracts from treated tissues were performed using 70-day post-immune rabbit serum. RESULTS: Immunoblot analysis of untreated BP or PAV with pre-immune serum showed no immune banding. The immune banding density increased progressively when immunoblots were performed with 14-day through 70-day post-immune serum. The immunoblot analysis of treated BP and PAV showed that soluble protein antigen removal from SDS- or SD-treated tissues was incomplete. CONCLUSION: Immunoblot analysis is a sensitive and robust assay for detecting soluble protein xenogeneic antigens after the decellularization of xenogeneic bioscaffolds. Under the study conditions, hypotonic lysis, SDS or SD detergent treatment, and aqueous wash-out-based decellularization of bovine pericardium and porcine aortic valve conduit did not completely remove detectable protein antigens.

Use of fat-fed rats to study the metabolic and vascular sequelae of obesity and beta-adrenergic antagonism.

Obesity-associated cardiovascular disease exerts profound human and monetary costs, creating a mounting need for cost-effective and relevant in vivo models of the complex metabolic and vascular interrelationships of obesity. Obesity is associated with endothelial dysfunction and inflammation. Free fatty acids (FFA), generated partly through beta-adrenergic receptor-mediated lipolysis, may impair endothelium-dependent vasodilation (EDV) by proinflammatory mechanisms. beta-Adrenergic antagonists protect against cardiovascular events by mechanisms not fully defined. We hypothesized that beta antagonists may exert beneficial effects, in part, by inhibiting lipolysis and reducing FFA. Further, we sought to evaluate the fat-fed rat as an in vivo model of obesity-induced inflammation and EDV. Control and fat-fed rats were given vehicle or beta antagonist for 28 d. Serum FFA were measured to determine the association to serum IL6, TNFalpha, and C-reactive protein and to femoral artery EDV. Compared with controls, fat-fed rats weighed more and had higher FFA, triglyceride, leptin, and insulin levels. Unexpectedly, in control and fat-fed rats, beta antagonism increased FFA, yet inflammatory cytokines were reduced and EDV was preserved. Therefore, reduction of FFA is unlikely to be the mechanism by which beta antagonists protect the endothelium. These results reflect the need for validation of ex vivo models of obesity-induced inflammation and endothelial dysfunction, concurrent with careful control of dietary fat composition and treatment duration.

Autocrine serotonin and transforming growth factor beta 1 signaling mediates spontaneous myxomatous mitral valve disease.

BACKGROUND AND AIM OF THE STUDY: Although serotonin and serotoninergic drugs are known to cause myxomatous-like valvulopathy, the role of serotonin in spontaneous myxomatous valve disease (MVD) remains unclear. Tryptophan hydroxylase 1 (TPH1) is the limiting enzyme for peripheral serotonin synthesis, and its expression in myxomatous valves could implicate an autocrine serotonin signaling mechanism. Studies in cultured cells demonstrate a close coupling between serotonin and transforming growth factor beta1 (TGFbeta1) signaling. The study aim was to investigate serotonin and TGFbeta1 signaling in spontaneous MVD. METHODS: In canine normal and myxomatous mitral valves, target signaling proteins including TPH1, serotonin 2B receptor (5HT(2B)R), serotonin transmembrane transporter (SERT), total and phosphorylated extracellular signaling-regulated kinase (ERK) 1/2, latent TGFbeta1 and TGFbeta1 receptors I and II, were studied using immunohistochemistry and immunoblot analysis. In human myxomatous valves, TPH1 was determined using immunofluorescence and immunoblot analysis. RESULTS: In canine mitral valves, both 5HT(2B)R and TPH1 were increased in myxomatous valves, whereas SERT, a key protein in serotonin metabolism, was decreased in myxomatous valves. Phosphorylated, but not total, ERK 1/2 was increased in myxomatous valves, consistent with an enhanced active serotonin signaling. The expression of TGFbeta1 receptors I and II, and of latent TGFbeta1, was increased in myxomatous valves. Human myxomatous mitral valves expressed TPH1. CONCLUSION: The expression of TPH1 by canine and human myxomatous valves demonstrates a capacity for local serotonin production. Key signaling protein expression patterns support active serotonin and TGFbeta1 signaling in canine myxomatous valves. These findings implicate an autocrine serotonin and TGFbeta1 mechanism in the pathogenesis of spontaneous MVD.

Does sodium dodecyl sulfate wash out of detergent-treated bovine pericardium at cytotoxic concentrations?

BACKGROUND AND AIM OF THE STUDY: The ionic detergent sodium dodecyl sulfate (SDS) is a proposed treatment for the removal of antigenic proteins from unfixed biological scaffolds used in tissue engineering. However, questions remain about possible cytotoxic effects of SDS-treated tissues. The study aims were to: (i) develop a sensitive SDS assay for physiological solutions; (ii) measure SDS concentrations in the washing media of SDS-treated tissue; and (iii) determine cytotoxic SDS concentrations in cultured ovine vascular cells. METHODS: An assay was developed to monitor SDS concentrations at microM levels, based on attenuated total reflectance infrared spectroscopy. Bovine pericardium was treated with SDS (1.0 to 0.01%) and washed for 96 h. The SDS concentration in the washing media was measured at 24-h intervals; data were expressed as microM/g tissue. Ovine vascular cells were cultured in DME media at 37 degrees C for 48 h in various SDS concentrations (10 to 1000 microM). The cells were then counted, and the percentage live cells expressed, based on trypan blue exclusion (n=5). RESULTS: SDS concentrations > or =10 microM significantly reduced (p < 0.05) the total cell number, while concentrations > or =100 microM reduced (p < 0.05) the percentage live cells of ovine vascular cell cultures. SDS was present in the washing media of SDS-treated bovine pericardium. SDS leaching from bovine pericardium was found to depend on the SDS concentration used for the treatment, and diminished with time. CONCLUSION: SDS leaches from SDS-treated bovine pericardium at concentrations that are potentially cytotoxic. An understanding of the dynamics of SDS washout, based on a sensitive SDS assay, may lead to the creation of protocols for the preparation of biological scaffolds that are free from cytotoxic leaching.

Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs.

Valvular heart disease accounts for over 20 000 deaths and 90 000 hospitalizations yearly in the United States. Myxomatous valve disease (MVD) is the most common disease of the mitral valve in humans and dogs. MVD is pathologically identical in these species and its pathogenesis is poorly understood. The objectives of this study were to (i) develop proteomic methodology suitable for analysis of extracellular matrix-rich heart valve tissues and (ii) survey over- and under-expressed proteins that could provide mechanistic clues into the pathogenesis of MVD. Normal, early-stage, and late-stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis was used to quantify differential protein expression. Proteins were classified by function and clustered according to differential expression patterns. More than 300 proteins, with 117 of those being differentially expressed, were identified. Hierarchical sample clustering of differential protein profiles showed that early- and late-stage valves were closely related. This finding suggests that proteome changes occur in early degeneration stages and these persist in late stages, characterizing a diseased proteome that is distinct from normal. Shotgun proteome analysis of matrix-rich canine heart valves is feasible, and should be applicable to human heart valves. This study provides a basis for future investigations into the pathogenesis of MVD.