Evaluation of right ventricular diastolic function, systolic function, and circulating galectin-3 concentrations in dogs with pulmonary stenosis.

BACKGROUND: Cardiovascular diseases with increased right ventricular (RV) afterload induce RV diastolic and systolic dysfunction, and myocardial fibrosis in humans. Studies in dogs with pulmonary stenosis (PS) evaluating RV diastolic function and markers of myocardial fibrosis are lacking. HYPOTHESIS/OBJECTIVES: Dogs with PS have echocardiographic evidence of RV diastolic and systolic dysfunction and increased serum concentrations of galectin-3 (Gal-3), a surrogate biomarker for myocardial fibrosis. ANIMALS: Forty client-owned dogs (10 controls, 30 with PS). METHODS: Prospective study. All dogs had systemic blood pressure measurement, serum biochemical analysis, echocardiography, and measurement of serum Gal-3 concentration performed. RESULTS: Variables of RV diastolic function were obtained in 39/40 dogs. Trans-tricuspid flow velocity in early diastole to trans-tricuspid flow velocity in late diastole ratios (RV E/A) were lower (P < .001) in dogs with PS (median, 0.94; range, 0.62-2.04) compared to controls (1.78; 1.17-2.35). Trans-tricuspid flow velocity in early diastole to tricuspid annular myocardial velocity in early diastole ratios (RV E/e') were higher (P < .001) in dogs with PS (11.55; 4.69-28) compared to control (6.21; 5.16-7.21). Variables of RV systolic function were lower in dogs with PS (P = <.001). Serum Gal-3 concentration was higher (P = .002) in dogs with PS (285.1 pg/mL; 94.71-406.97) compared to control dogs (162.83 pg/mL; 52.3-232.82). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with PS have RV diastolic and systolic dysfunction, and increased Gal-3 concentrations. These findings suggest the presence of RV myocardial fibrosis in dogs with PS, which could impact clinical management.

Utility of cardiac MRI to diagnose myocardial ischemia and fibrosis in dogs with cardiomegaly secondary to myxomatous mitral valve disease.

OBJECTIVE: To assess whether cardiac MRI or various biomarkers can be used to detect myocardial ischemia and fibrosis in dogs with cardiomegaly secondary to myxomatous mitral valve disease (MMVD). ANIMALS: 6 dogs with cardiomegaly secondary to naturally occurring stage B2 MMVD being treated only with pimobendan with or without enalapril and 6 control dogs with no cardiac disease. All dogs were ≥ 5 years old with no systemic illness. PROCEDURES: Serum cardiac troponin I and concentrations were measured, and dogs were anesthetized for cardiac MRI with ECG-triggered acquisition of native T1- and T2-weighted images. Gadolinium contrast was administered to evaluate myocardial perfusion and late gadolinium enhancement (LGE). Mean T1 and T2 values and regions of LGE were measured with dedicated software. Extracellular volume (ECV) was estimated on the basis of Hct and T1 values of myocardium and surrounding blood. Subjective analysis for myocardial perfusion deficits was performed. RESULTS: Dogs with MMVD had significantly (P = .013) higher cardiac troponin I concentrations than control dogs, but galectin-3 concentrations did not differ (P = .08) between groups. Myocardial fibrosis was detected in 4 dogs with MMVD and 3 control dogs; no dogs had obvious myocardial perfusion deficits. Native T1 and T2 values, postcontrast T1 values, and ECV values were not significantly different between groups (all P > .3). CLINICAL RELEVANCE: Results suggest that some dogs with cardiomegaly secondary to MMVD may not have clinically relevant myocardial fibrosis.

Effect of recombinant equine interleukin-1ß on function of equine endothelial colony-forming cells in vitro.

OBJECTIVE: To investigate the effects of recombinant equine IL-1ß on function of equine endothelial colony-forming cells (ECFCs) in vitro. SAMPLE: ECFCs derived from peripheral blood samples of 3 healthy adult geldings. PROCEDURES: Function testing was performed to assess in vitro wound healing, tubule formation, cell adhesion, and uptake of 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) by cultured ECFCs. Cell proliferation was determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Effects on function test results of different concentrations and exposure times of recombinant equine IL-1ß were assessed. RESULTS: Challenge of cultured ECFCs with IL-1ß for 48 hours inhibited tubule formation. Continuous challenge (54 hours) with IL-1ß in the wound healing assay reduced gap closure. The IL-1ß exposure did not significantly affect ECFC adhesion, DiI-Ac-LDL uptake, or ECFC proliferation. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggested a role for IL-1ß in the inhibition of ECFC function in vitro. Functional changes in ECFCs following challenge with IL-1ß did not appear to be due to changes in cell proliferative capacity. These findings have implications for designing microenvironments for and optimizing therapeutic effects of ECFCs used to treat ischemic diseases in horses.

Effects of pimobendan on left atrial transport function in cats.

BACKGROUND: Arterial thromboembolism is a sequela of hypertrophic cardiomyopathy (HCM) in cats related to left atrial (LA) enlargement and dysfunction. HYPOTHESIS: Pimobendan improves LA transport function in cats. ANIMALS: Twenty-two client-owned cats with HCM and 11 healthy cats. METHODS: Prospective, double-blind, randomized, placebo-controlled clinical cohort study. Cats were randomized to receive either pimobendan (0.25 mg/kg PO q12h) or placebo for 4 to 7 days. Nineteen echocardiographic variables of LA size and function were evaluated. Statistical comparisons included t tests, analysis of variance, and multivariable analyses. RESULTS: Peak velocity of left auricular appendage flow (LAapp peak; mean ± SD, 0.85 ± 0.20 vs 0.71 ± 0.22 m/s; P = .01), maximum LA volume (P = .03), LA total emptying volume (P = .03), peak velocity of late diastolic transmitral flow (A peak velocity; 0.77 ± 0.12 vs 0.62 ± 0.17 m/s; P = .05), and A velocity time integral (A VTI; 3.05 ± 0.69 vs 3.37 ± 0.49; P = .05) were increased after pimobendan. Mean change after pimobendan was larger in cats with HCM compared to healthy cats for LA fractional shortening (2.1% vs -2.1%; P = .05), A VTI (0.58 vs 0.01 cm; P = .01), LAapp peak (0.20 vs 0.02 m/s; P = .02), LA kinetic energy (3.51 vs -0.10 kdynes-cm; P = .05), and LA ejection force (1.93 vs -0.07 kdynes; P = .01) in the multivariable model. The stronger effect of pimobendan in cats with HCM was independent of LA size. CONCLUSIONS AND CLINICAL IMPORTANCE: We identified positive, albeit minor, effects of pimobendan on LA function in cats with HCM. Whether or not treatment with pimobendan decreases the risk of cardiogenic embolism deserves further study.

Acquired left-to-right shunting through a valve-incompetent foramen ovale in a cat with hypertrophic cardiomyopathy and congestive heart failure.

CASE SUMMARY: An 8-year-old spayed female domestic shorthair cat was presented for a recheck evaluation of hypertrophic cardiomyopathy and chronic kidney disease. Three years prior to presentation, the patient was diagnosed with obstructive hypertrophic cardiomyopathy and started on atenolol. The left ventricular outflow tract obstruction subsequently resolved. Biochemical analysis a week prior to presentation demonstrated severe azotemia. Transthoracic echocardiograph revealed pericardial effusion, pleural effusion, severe left ventricular concentric hypertrophy, severe left atrial enlargement and continuous left-to-right flow through the interatrial septum near the fossa ovalis. The patient was euthanized owing to poor prognosis, and gross examination at necropsy revealed a valve-incompetent patent foramen ovale secondary to severe left atrial dilation. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first report of an acquired left-to-right shunt through a valve-incompetent foramen ovale in a cat with hypertrophic cardiomyopathy. Severe left atrial dilation was suspected to cause interatrial shunting through the valve-incompetent foramen ovale, and this finding may be relevant to echocardiographic evaluations in other cats.

Pharmacokinetics of Pimobendan and Its Metabolite O-Desmethyl-Pimobendan Following Rectal Administration to Healthy Dogs.

Objective: This study describes the pharmacokinetics of parent pimobendan (PIM) and its active metabolite, o-desmethyl-pimobendan (ODMP), after oral and rectal administration of pimobendan to healthy dogs. Animals: A total of eight healthy privately owned dogs were used in this study. Procedures: The dogs received a single dose (0.5 mg/kg) of a commercially available pimobendan tablet per os (PO). Twelve blood samples were collected over a 12-h period for pharmacokinetic analysis. After a 24-h washout period, the dogs received the same dose of pimobendan solution per rectum (PR), and samples were obtained at the same time for analysis. Results: For PIM, PO vs. PR, respectively, the mean maximum plasma concentration (C max, ng/ml) was 49.1 ± 28.7 vs. 10.1 ± 2, the time to reach a maximum concentration (T max, h) was 2.1 ± 0.9 vs. 1 ± 0.4, the disappearance half-life (t 1/2, h) was 1.8 ± 0.8 vs. 2.2 ± 0.6, and the area under the concentration-time curve (AUC, ng*h/ml) was 148.4 ± 71.6 vs. 31.1 ± 11.9, with relative bioavailability (F, %) of 25 ± 8. For ODMP, PO vs. PR, respectively, C max was 30.9 ± 10.4 vs. 8.8 ± 4.8, T max was 3.2 ± 1.6 vs. 1.7 ± 1.1, and t 1/2 was 5.0 ± 2.7 vs. 8.3 ± 4.8, with AUC of 167.8 ± 36.2 vs. 50.1 ± 19.2 and F of 28 ± 6. The differences between PO and PR were significant (P < 0.03) for AUC and C max for both PIM and ODMP. Conclusions and Clinical Relevance: The pharmacokinetics of PIM and ODMP were described following PO and PR administration. The findings suggest that pimobendan PR might achieve effective concentrations and, as such, warrant future studies of clinical effectiveness in treating dogs with congestive heart failure and which are unable to receive medication PO.

Development of Spontaneous Echocardiographic Contrast After Transarterial Occlusion of a Patent Ductus Arteriosus in an Adult Dog With Concurrent Pulmonary Hypertension.

An 8-year-old intact female Chihuahua was presented for evaluation and possible occlusion of a previously diagnosed patent ductus arteriosus (PDA). Transthoracic echocardiography revealed left ventricular and left atrial enlargement, enlargement of the main pulmonary artery, and a PDA with bidirectional shunting. Tricuspid regurgitant velocities suggested moderate pulmonary hypertension. The PDA was occluded with an Amplatz® Canine Duct Occluder using a transarterial approach on the following day. No immediate complications were observed other than an acute decrease in left ventricular systolic function. One day after the PDA occlusion transthoracic echocardiography revealed no residual ductal flow, but there was spontaneous echocardiographic contrast in the left ventricle. The patient was discharged with sildenafil, pimobendan, and clopidogrel. Five weeks later when the patient was presented for a recheck examination, the previously documented spontaneous echocardiographic contrast was no longer present. Finding spontaneous echocardiographic contrast in the dog has not previously been reported in association with PDA occlusion.

Cell engraftment, vascularization, and inflammation after treatment of equine distal limb wounds with endothelial colony forming cells encapsulated within hydrogel microspheres.

BACKGROUND: Endothelial colony forming cells (ECFCs) may be useful therapeutically in conditions with poor blood supply, such as distal limb wounds in the horse. Encapsulation of ECFCs into injectable hydrogel microspheres may ensure cell survival and cell localization to improve neovascularization and healing. Autologous ECFCs were isolated from 6 horses, labeled with quantum nanodots (QD), and a subset were encapsulated in poly(ethylene) glycol fibrinogen microspheres (PEG-Fb MS). Full-thickness dermal wounds were created on each distal limb and injected with empty PEG-Fb MS, serum, ECFCs, or ECFCs encapsulated into PEG- Fb MS (ECFC/MS). Analysis included wound surface area (WSA), granulation tissue scoring (GS), thermography, collagen density staining, and immunohistochemical staining for endothelial and inflammatory cells. The purpose of this study was to track cell location and evaluate wound vascularization and inflammatory response after injection of ECFC/MS or naked ECFCs in equine distal limb wounds. RESULTS: ECFCs were found near and within newly formed blood vessels up to 3 weeks after injection. ECFC and ECFC/MS groups had the greatest blood vessel quantity at week 1 in the wound periphery. Wounds treated with ECFCs and ECFC/MS had the lowest density of neutrophils and macrophages at week 4. There were no significant effects of ECFC or ECFC/MS treatment on other measured parameters. CONCLUSIONS: Injection of microsphere encapsulated ECFCs was practical for clinical use and well-tolerated. The positive ECFC treatment effects on blood vessel density and wound inflammation warrant further investigation.

Pharmacokinetics of a commercially available product and a compounded formulation of extended-release levetiracetam after oral administration of a single dose in cats.

OBJECTIVE: To compare pharmacokinetics of levetiracetam in serum and CSF of cats after oral administration of extended-release (ER) levetiracetam. ANIMALS: 9 healthy cats. PROCEDURES: Cats received 1 dose of a commercially available ER levetiracetam product (500 mg, PO). Thirteen blood and 10 CSF samples were collected over a 24-hour period for pharmacokinetic analysis. After 1 week, cats received 1 dose of a compounded ER levetiracetam formulation (500 mg, PO), and samples were obtained at the same times for analysis. RESULTS: CSF concentrations of levetiracetam closely paralleled serum concentrations. There were significant differences between the commercially available product and the compounded formulation for mean ± SD serum maximum concentration (Cmax; 126 ± 33 µg/mL and 169 ± 51 µg/mL, respectively), Cmax corrected for dose (0.83 ± 0.10 µg/mL/mg and 1.10 ± 0.28 µg/mL/mg, respectively), and time to Cmax (5.1 ± 1.6 hours and 3.1 ± 1.5 hours, respectively). Half-life for the commercially available product and compounded formulation of ER levetiracetam was 4.3 ± 2.0 hours and 5.0 ± 1.6 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The commercially available product and compounded formulation of ER levetiracetam both maintained concentrations in healthy cats 12 hours after oral administration that have been found to be therapeutic in humans (ie, 5 µg/mL). Results of this study supported dosing intervals of 12 hours, and potentially 24 hours, for oral administration of ER levetiracetam to cats. Monitoring of serum concentrations of levetiracetam can be used as an accurate representation of levetiracetam concentrations in CSF of cats.

A prospective, randomized, double-blind, placebo-controlled pilot study of sacubitril/valsartan (Entresto) in dogs with cardiomegaly secondary to myxomatous mitral valve disease.

BACKGROUND: The effects of sacubitril/valsartan (S/V) on the renin-angiotensin-aldosterone system (RAAS) in dogs with cardiomegaly secondary to myxomatous mitral valve disease (MMVD) are currently unknown. OBJECTIVES: To determine the pharmacodynamic effects of S/V on the RAAS, natriuretic peptide concentrations, systolic arterial pressure (SAP), tests of renal function, and serum electrolyte concentrations in dogs with cardiomegaly secondary to MMVD. ANIMALS: Thirteen client-owned dogs weighing 4-15 kg with American College of Veterinary Internal Medicine (ACVIM) Stage B2 MMVD. METHODS: Prospective, randomized, double-blind, placebo-controlled pilot study of S/V in dogs with ACVIM Stage B2 MMVD. RESULTS: Thirteen dogs were recruited: S/V (n = 7) and placebo (n = 6). The median percentage increase in urinary aldosterone to creatinine ratio (UAldo : C) between day 0 and day 30 was significantly lower in the S/V group (12%; P = .032) as compared with the placebo group (195%). The median percentage decrease of NT-proBNP concentration from day 0 to day 30 was not statistically different between groups (P = .68). No statistical differences were seen in echocardiographic, thoracic radiographic, SAP, or serum biochemical test results measured at any time point between groups. No adverse events were observed for dogs in either group. CONCLUSION AND CLINICAL IMPORTANCE: Sacubitril/valsartan may provide a new pharmaceutical method to effectively inhibit the RAAS in dogs with ACVIM Stage B2 MMVD.

Growth and function of equine endothelial colony forming cells labeled with semiconductor quantum dots.

BACKGROUND: Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair in vivo and are attractive for clinical use in ischemic disease. Tracking of stem and progenitor cells is essential to determine engraftment after administration. Semiconductor quantum dots (QD) are promising for cell labeling due to their ease of uptake by many cell lines and their continued presence after many cell generations. The purpose of this study was to evaluate function and growth of equine EPCs after QD labeling. Additionally, this study evaluated the duration of QD label retention and mechanisms of QD label loss. RESULTS: Endothelial colony forming cells (ECFCs) from adult horses (N = 3) were employed for this study, with QD labeled and unlabeled ECFCs tested from each horse. Cell proliferation of ECFCs labeled with QD at 20 nM was quantified by comparing the number of cell doublings per day (NCD) and the population doubling time (PDT) in labeled and unlabeled cells. Function of labeled and unlabeled ECFCs was assessed by comparing uptake of acetylated low-density lipoprotein (DiO-Ac-LDL) and tubule formation on growth factor containing matrix. Cell proliferation was not impacted by QD labeling; both NCD (p = 0. 95) and PDT (P = 0. 91) did not differ between unlabeled and QD labeled cells. Function of ECFCs assessed by DiO-Ac-LDL and tubule formation was also not different between unlabeled and QD labeled cells (P = 0. 33 and P = 0. 52, respectively). ECFCs retained their QD labeling over 7 passages with both 5 nM and 20 nM label concentrations. Reduction in label intensity was observed over time, and the mechanism was determined to be cell division. CONCLUSIONS: Equine ECFCs are effectively labeled with QD, and QD concentrations up to 20 nM do not affect cell growth or function. QD label loss is a result of cell division. The use of QD labeling with equine EPCs may be an ideal way to track engraftment of EPCs for in vivo applications.

Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and ß subunits separately (TSD α + ß) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + ß sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + ß), and ganglioside clearance was most widespread in the TSD α + ß high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

Effect of heartworm disease and heartworm-associated respiratory disease (HARD) on the right ventricle of cats.

BACKGROUND: Dirofilaria immitis infection occurs in dogs and cats, both of which species are clinically affected by mature adult infections. Cats are uniquely affected by immature-adult infections with an inflammatory pulmonary disease called Heartworm-Associated Respiratory Disease (HARD). D. immitis infection causes pulmonary parenchymal and vascular pathology in the dog and cat. Dogs develop pulmonary hypertension and cor pulmonale, whereas the development of pulmonary hypertension is rare in the cat. D. immitis infection in the dog causes alteration of the right ventricular (RV) extracellular matrix, including a decrease in myocardial collagen. In this study, the RV myocardial changes of cats infected with adult and immature-adult D. immitis were assessed. METHODS: The cardiopulmonary systems of six groups of SPF cats (n = 9-10 per group) were examined 8 or 18 months after infection with L3 D. immitis. Two groups were untreated and allowed to develop adult HW; two groups were treated with ivermectin starting 3 months post infection, thus allowing HARD but no mature adult heartworms; and two groups were treated with selamectin beginning 1 month post infection, preventing development of L5 or adult heartworms. A group of specific pathogen free (SPF) normal cats was utilized as a negative control (n = 12). Lung pathologic lesions were objectively assessed, and both RV and left ventricular (LV) weights were obtained to calculate an RV/LV ratio. Intramural RV myocardial collagen content was quantitatively assessed. RESULTS: RV/LV weight ratios were not different between groups. Negative control cats had significantly greater RV collagen content than all other affected groups (P = 0.032). Analysis of the RV/LV ratios and collagen content revealed no significant relationship (r = 0.03, P = 0.723, respectively). Collagen content had a modest, but significant, negative correlation, however, with both pulmonary vascular pathology (r = -0.25, P = 0.032) as well as the total pulmonary parenchymal and vascular pathology (r = -0.26, P = 0.025). CONCLUSIONS: Cats infected with mature and immature D. immitis did not develop RV hypertrophy but did demonstrate loss of RV myocardial collagen content. The collagen loss was present at 8 and 18 months after infection in all infected cats. This loss of RV myocardial collagen was correlated with the severity of pulmonary parenchymal and vascular pathology.

Encapsulation of Equine Endothelial Colony Forming Cells in Highly Uniform, Injectable Hydrogel Microspheres for Local Cell Delivery.

A common challenge in cell therapy is the inability to routinely maintain survival and localization of injected therapeutic cells. Delivering cells by direct injection increases the flexibility of clinical applications, but may cause low cell viability and retention rates due to the high shear forces in the needle and mechanical wash out. In this study, we encapsulated endothelial colony forming cells (ECFCs) in poly(ethylene glycol)-fibrinogen (PF) hydrogel microspheres using a custom-built microfluidic device; this system supports rapid encapsulation of high cell concentrations (10 million cells per mL) and resulting cell-laden microspheres are highly uniform in shape and size. The encapsulated ECFCs were shown to have >95% viability and continued to rapidly proliferate. Expression of cell markers (von Willebrand factor, CD105, and CD14), the ability to form tubules on basement membrane matrix, and the ability to take up low-density lipoprotein were similar between pre- and post-encapsulated cells. Viability of encapsulated ECFCs was maintained after shear through 18-23-gauge needles. Ex vivo and in vivo cell delivery studies were performed by encapsulating and injecting autologous equine ECFCs subcutaneously into distal limb full-thickness wounds of adult horses. Injected ECFCs were visualized by labeling with fluorescent nanodots before encapsulation. One week after injection, confocal microscopy analysis of biopsies of the leading edges of the wounds showed that the encapsulated ECFCs migrated into the surrounding host tissue indicating successful retention and survival of the delivered ECFCs. Rapid, scalable cell encapsulation into PF microspheres was demonstrated to be practical for use in large animal cell therapy and is a clinically relevant method to maintain cell retention and survival after local injection.