手嶋 隆洋

Introduction The therapeutic efficacy of mesenchymal stromal cells (MSCs) in inflammatory bowel disease is not completely known and is not consistent. Priming with inflammatory cytokines has been proposed to adapt MSCs to an inflammatory environment to have them ready to counteract it, but may have undesirable effects on MSCs, such as increased immunogenicity. In this study, we hypothesized that priming MSCs with inflamed intestinal tissue would more effectively enhance their therapeutic effect on intestinal inflammation. Methods The capacity of canine adipose-derived MSCs (cADSCs) primed with colon tissue homogenates from mice with experimentally induced colitis or a combination of tumor necrosis factor-α and interferon-γ to inhibit T-cell proliferation was analyzed, along with their own apoptosis, proliferation, cell surface marker expression, and transcriptome. In addition, colitis mice were treated with the primed cADSCs to assess colitis severity and immune cell profile. Results Priming with cytokines induced apoptosis, decreased cell proliferation, and major histocompatibility complex-II gene expression in cADSCs, but these adverse effects were mild or absent with colitis-tissue priming. cADSCs primed with colitis tissue reduced the severity of colitis via the induction of M2 macrophages and T-regulatory cells and suppression of T-helper (Th)1/Th17-cell responses, and their effects were comparable to those of cytokine-primed cells. Discussion Our results emphasize the importance of the activation of MSCs by the appropriate microenvironment to maximize their therapeutic effect.

Inflammatory bowel disease (IBD) is a major subtype of chronic enteropathies in dogs and cats. Conventional drugs such as immunomodulatory medicines as glucocorticoids and/or other anti-inflammatory are mainly applied for treatment. However, these drugs are not always effective to maintain remission from IBD and are limited by unacceptable side effects. Hence, more effective and safe therapeutic options need to be developed. Mesenchymal stem cells (MSCs) are multipotent stem cells with a self-renewal capacity, and have immunomodulatory, anti-inflammatory, anti-fibrotic, and tissue repair properties. Therefore, the application of MSCs as an alternative therapy for IBD has great potential in veterinary medicine. The efficacy of adipose tissue-derived MSC (ADSC) therapy for IBD in dogs and cats has been reported, including numerous studies in animal models. However, treatment outcomes in clinical trials of human IBD patients have not been consistent with preclinical studies. MSC-based therapy for various diseases has received widespread attention, but various problems in such therapy remain, among which no consensus has been reached on the preparation and treatment procedures for MSCs, and cellular heterogeneity of MSCs may be an issue. This review describes the current status of ADSC therapy for canine and feline IBD and summarizes the cellular heterogeneity of canine ADSCs, to highlight the necessity for further reduction or elimination of MSCs heterogeneity and standardization of MSC-based therapies.

BACKGROUND: Pulmonary hypertension (PH) secondary to respiratory disease is caused by pulmonary vascular remodeling and hypoxia. Severe PH can induce various clinical signs, including syncope and right-sided heart failure. HYPOTHESIS/OBJECTIVES: To investigate the echocardiographic characteristics in dogs with PH secondary to respiratory diseases. ANIMALS: Thirty-one dogs with respiratory diseases with or without PH and 15 healthy dogs. METHODS: Prospective cross-sectional study. Dogs were classified according to respiratory disease (obstructive airway/lung disease [OALD] or restrictive lung disease [RLD]) and PH-relevant signs. The association between echocardiographic variables and PH (classified by respiratory disease and PH-relevant signs) was investigated. RESULTS: Twenty-one dogs were diagnosed with PH; of these, 11 showed PH-related signs (OALD, n = 2; RLD, n = 9), 14 had right ventricular hypertrophy, and 19 had pulmonary arterial enlargement. Right ventricular dysfunction and dilatation were observed only in dogs with PH-related signs (n = 10). Left and right ventricular stroke volumes were significantly lower in dogs with PH (median [interquartile range]: 17.2 [12.4-20.8] and 16.8 [15.3-29.5] mL/m2 , respectively). Dogs with RLD had higher echocardiography-estimated pulmonary vascular resistance than those with OALD (median [interquartile range]: 3.1 [1.9-3.3] and 1.6 [1.3-2.2], respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Pulmonary arterial enlargement was the most common echocardiographic finding in dogs with PH secondary to respiratory diseases. Right ventricular dysfunction, dilatation, and decreased left and right ventricular stroke volume were significantly associated with the PH-related signs, indicating that comprehensive echocardiography is recommended in dogs with respiratory disease. Restricted lung disease might induce more severe PH than OALD.

Pulmonary hypertension (PH) is a life-threatening complication in dogs with cardiopulmonary disease. Epoprostenol is an intravenous pulmonary vasodilator used to treat PH in humans; however, its efficacy in dogs remains unknown. We investigated the cardiovascular effects of epoprostenol and several cardiac agents for acute heart failure in canine models of chronic PH. Six dogs with chronic PH were anesthetized and underwent right heart catheterization and echocardiography before and after infusion of epoprostenol, dobutamine, dopamine and pimobendane. (The drug administration order was the same for all dogs). High-dose epoprostenol (15-20 ng/kg/min) tended to decrease pulmonary arterial pressure (PAP) while significantly decreasing pulmonary and systemic vascular resistance and increasing left and right ventricular (LV and RV, respectively) function. Pimobendan significantly increased LV and RV functions without increasing PAP. Conversely, dobutamine and dopamine significantly increased LV and RV function as well as PAP. This study revealed the efficacy of epoprostenol in treating canine PH through its pulmonary and systemic vasodilating effects. Although catecholamines improve LV and RV function, they might worsen PH pathophysiology, and careful monitoring may be necessary when using these drugs. Pimobendan improved LV and RV function without increasing PAP; however, a stronger vasodilating effect was observed with epoprostenol.

Mesenchymal stem cells are expected to be a cell source for stem cell therapy of various diseases in veterinary medicine. However, donor-dependent cell heterogenicity has been a cause of inconsistent therapeutic efficiency. Therefore, we established immortalized cells from canine adipose tissue-derived mesenchymal stem cells (ADSCs) to minimize cellular heterogeneity by reducing the number of donors, evaluated their properties, and compared them to the primary cells with RNA-sequencing. Immortalized canine ADSCs were established by transduction with combinations of the R24C mutation of human cyclin-dependent kinase 4 (CDKR24C), canine cyclin D1, and canine TERT. The ADSCs transduced with CDK4R24C, cyclin D1, and TERT (ADSC-K4DT) or with CDK4R24C and cyclin D1 (ADSC-K4D) showed a dramatic increase in proliferation (population doubling level >100) without cellular senescence compared to the primary ADSCs. The cell surface markers, except for CD90 of the ADSC-K4DT and ADSC-K4D cells, were similar to those of the primary ADSCs. The ADSC-K4DT and ADSC-K4D cells maintained their trilineage differentiation capacity and chromosome condition, and did not have a tumorigenic development. The ability to inhibit lymphocyte proliferation by the ADSC-K4D cells was enhanced compared with the primary ADSCs and ADSC-K4DT cells. The pathway analysis based on RNA-sequencing revealed changes in the pathways mainly related to the cell cycle and telomerase. The ADSC-K4DT and ADSC-K4D cells had decreased CD90 expression, but there were no obvious defects associated with the decreased CD90 expression in this study. Our results suggest that ADSC-K4DT and ADSC-K4D cells are a potential novel cell source for mesenchymal stem cell therapy.

BACKGROUND: Progression to combined post- and pre-capillary pulmonary hypertension (PH) provides prognostic information in human patients with post-capillary PH. Pulmonary vascular resistance estimated by echocardiography (PVRecho) is useful for the stratification of dogs with myxomatous mitral valve disease (MMVD) and detectable tricuspid regurgitation. OBJECTIVES: To evaluate the prognostic value of PVRecho in dogs with MMVD. ANIMALS: Fifty-four dogs with MMVD and detectable tricuspid regurgitation. METHODS: Prospective cohort study. All dogs underwent echocardiography. The PVRecho was calculated based on tricuspid regurgitation and the velocity-time integral of the pulmonary artery flow. To evaluate the influence of echocardiographic variables on cardiac-related deaths, Cox proportional hazard analysis was performed. Additionally, Kaplan-Meier curves classified by PVRecho tertiles were made and compared using log-rank tests to evaluate the influence of PVRecho on all-cause mortality and cardiac-related death. RESULTS: The median follow-up time was 579 days. Forty-one dogs with MMVD (PH severity [number]: no or mild, 21/33; moderate, 11/11; severe, 9/10) died during the study. In the multivariable Cox proportional hazard analysis adjusted for age, sildenafil administration, and American College of Veterinary Internal Medicine stage of MMVD, left atrial to aortic diameter ratio and PVRecho remained significant (adjusted hazard ratio [95% confidence interval]: 1.2 [1.1-1.3] and 2.1 [1.6-3.0], respectively). Higher PVRecho showed a significant association with lower survival rates. CONCLUSIONS AND CLINICAL IMPORTANCE: Left atrial enlargement and high PVRecho were independent prognostic factors in dogs with MMVD and detectable tricuspid regurgitation.

In recent years, hypertrophic cardiomyopathy (HCM) in cats has become much more common in clinical practice due to improvements in diagnostic techniques and equipment performance. One phenotype is obstructive HCM with left ventricular (LV) outflow tract obstruction (DLVOTO). It has been reported that the presence or absence of DLVOTO does not affect long-term prognosis in cats with HCM. In this study, we evaluated and compared myocardial function in HCM-affected cats with and without DLVOTO using the two-dimensional speckle-tracking echocardiography. LV longitudinal strain of the endocardial, epicardial, and whole layer and LV circumferential strain of the epicardium were significantly decreased in all HCM-affected cats compared to healthy cats. However, these values were not significantly different between those with and without DLVOTO. In contrast, the endocardial and whole layers of LV circumferential strain were only significantly decreased in HCM-affected cats with DLVOTO compared to healthy cats. This could be attributed to the fact that the LV pressure load associated with DLVOTO affected the endocardial myocardium more in the LV endocardial layer, and that lower values of LV endocardial strain lowered the values of LV strain in the whole layer. In conclusion, our results suggest that LV myocardial function may have been more compromised in the HCM-affected cats with DLVOTO.

Mesenchymal stem cells (MSC) are currently being investigated for their therapeutic applications in a wide range of diseases. Although many studies examined peripheral venous administration of MSC, few have investigated the detailed intravenous administration procedures of MSC from their preparation until they enter the body. The current study therefore aimed to explore the most efficient infusion procedure for MSC delivery by preparing and infusing them under various conditions. Canine adipose-derived mesenchymal stem cells (cADSC) were infused using different infusion apparatuses, suspension solutions, allogenic serum supplementation, infusion time and rates, and cell densities, respectively. Live and dead cell counts were then assessed by manual measurements and flow cytometry. Efficiency of live- and dead-cell infusion and cell viability were calculated from the measured cell counts and compared under each condition. Efficiency of live-cell infusion differed significantly according to the infusion apparatus, infusion rate, and combination of cell density and serum supplementation. Cell viability after infusion differed significantly between the infusion apparatuses. The optimal infusion procedure resulting in the highest cell delivery and viability involved suspending cADSC in normal saline supplemented with 5% allogenic serum at a density of 5 × 105 cells/mL, and infusing them using an automatic infusion device for 15 min. This procedure is therefore recommended as the standard procedure for the intravenous administration of ADSC in terms of cell-delivery efficiency.

An 11-year-old intact female Papillion weighing 2.1 kg was referred to our institution with the main complaint of shallow, rapid breathing. At the first visit (day 0), although clinical signs improved due to the use of medication from the primary hospital, transthoracic radiography and echocardiography revealed left heart enlargement and left ventricular dysfunction. A clinical diagnosis of dilated cardiomyopathy (DCM) was made and oral administration of pimobendan, temocapril, and taurine was initiated. However, on day 10, the respiratory status worsened and furosemide was prescribed. On day 54, no significant improvement in heart size was observed. Additionally, the diet that this patient received met the recommendation for diet-related DCM by the U.S. Food and Drug Administration, and the patient's diet was changed from a grain-free diet to a grain-containing diet. On day 1191, the patient's respiratory status was stable and no clinical signs were observed. Transthoracic radiography and echocardiography revealed an improvement in left heart size. Additionally, improvements in the left and right ventricular myocardial strains were observed after changing the diet. We suggest that it may be necessary to suspect a dietary association with dilated cardiomyopathy, and a good prognosis might be expected by dietary modification, even in small-breed dogs.

Pulmonary hypertension (PH) is a fatal condition that affects many dogs. In humans, PH is often treated with beraprost sodium (BPS). However, the effectiveness of BPS for canine PH has not been established. This study aimed to evaluate the clinical and cardiovascular response of BPS in canine patients with PH of various causes. Sixteen dogs with PH (post-capillary PH, n = 8; pre-capillary PH, n = 8) were included. BPS was continuously administered twice daily at 15 µg/kg. All dogs underwent echocardiography, including speckle-tracking analysis and blood pressure measurement, before and after BPS administration. Continuous BPS administration (range: 13.2-22.0 µg/kg) significantly decreased the pulmonary and systemic vascular impedance and increased left and right ventricular myocardial strain. In dogs with post-capillary PH, BPS administration caused no significant worsening of the left atrial pressure indicators. No side effects of BPS were observed in any dog. BPS also improved cardiac function and pulmonary circulation through pulmonary vasodilation, suggesting that BPS may be an additional treatment option for canine PH of various causes. Particularly, BPS increased left ventricular function and systemic circulation without worsening the left heart loading condition in dogs with post-capillary PH.

Mesenchymal stem cells (MSCs) have excellent anti-inflammatory and immunomodulatory capabilities and therapeutic effects in some viral diseases. The therapeutic impact of MSCs mainly relies on the paracrine effects of various secreted substances. Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV1) are common and highly prevalent pathogens causing upper respiratory diseases, and FCV is associated with gingivostomatitis in cats. Recently, feline MSC treatment has been reported to improve the clinical symptoms of feline chronic gingivostomatitis, but the antiviral effects of feline MSCs on FCV and FHV1 are not known. In this study, we evaluated the antiviral efficacy of using feline MSC secretome as a conditioned medium on FCV and FHV1 viral replication in Crandell-Reese feline kidney (CRFK) cells, and RNA sequencing was used to analyze how the CRFK cells were altered by the MSC secretomes. The feline MSC secretome did not inhibit FCV or FHV1 viral entry into the CRFK cells but had antiviral effects on the replication of both FCV and FHV1 in a dose-dependent manner.

BACKGROUND: Right ventricular (RV) functional assessment has received considerable attention in veterinary medicine since various diseases, such as cardiovascular, respiratory, endocrine, and neoplastic disease, may affect RV function. Heart rate (HR) is an important factor that can influence RV function through changes in loading condition and contractility. However, no study has yet evaluated the association between HR and RV function in the same individuals. This study aimed to evaluate the influence of elevated HR on RV function using right heart catheterization and echocardiography, and investigate the association between right heart catheterization and echocardiographic indices. RESULTS: Right atrial pacing was performed in eight dogs at 120, 140, 160, and 180 bpm. With an increase in HR, the RV systolic volume, RV diastolic volume, and stroke volume significantly decreased; however, the cardiac output, end-systolic elastance (Ees), and effective arterial elastance (Ea) significantly increased. Significant changes were not observed in RV pressure and Ees/Ea. The RV area normalized by body weight, RV fractional area change normalized by body weight (RV FACn), and tricuspid annular plane systolic excursion normalized by body weight (TAPSEn) significantly decreased with increased HR. Peak systolic myocardial velocity of the lateral tricuspid annulus (RV s'), RV strain, and RV strain rate of only the RV free wall analysis (RV-SrL3seg) showed no significant changes with the increase in HR; however, there was an increase in the RV strain rate of the RV global analysis (RV-SrL6seg). Multiple regression analysis revealed that HR, RV FACn, and RV- SrL6seg had significant associations with the Ees, and the TAPSEn and RV-SrL3seg with Ees/Ea. CONCLUSIONS: Decreased venous return and shortened relaxation time decreased the RV FAC, TAPSE, RV s', and RV strain, and might underestimate the RV function. Ees increased with the increase in HR, reflecting the myocardial force-frequency relation; as a result, RV-SrL6seg could be a useful tool for Ees estimation. Additionally, the RV-SrL3seg could detect RV performance, reflecting the balance between RV contractility and RV afterload.

Real-time three-dimensional echocardiography (RT3DE) enables a noninvasive assessment of right ventricular (RV) morphology. However, no study has evaluated the relationship between RV function obtained by RT3DE and RV pressure-volume loops. This hypothesis-driven, experimental study aimed to assess the utility of RT3DE in the evaluation of RV morphology and function. Ten anesthetized beagle dogs sequentially underwent dobutamine infusion, acute infusion of lactated Ringer’s solution, and furosemide administration to alter RV contractility and loading conditions. RV pressure-volume loop-derived hemodynamic measurements and echocardiography, including two-dimensional speckle-tracking echocardiography and RT3DE, were performed in each study protocol. Bland–Altman analysis showed strong agreement in RV volume, ejection fraction, and stroke volume obtained by right heart catheterization and RT3DE. Multiple regression analyses revealed that the peak myocardial velocity of the lateral tricuspid annulus (RV s’) and global RV longitudinal strain rate were significantly associated with end-systolic elastance (adjusted r2 = 0.66, p < 0.001). RV s’, RV free wall longitudinal strain, and RT3DE-derived stroke volume/end-systolic RV volume ratio were associated with RV pressure-volume loops-derived end-systolic/arterial elastance ratio (adjusted r2 = 0.34, p < 0.001). RT3DE could detect the changes in catheterization-derived RV volume with a strong agreement and might be useful in estimating RV-pulmonary arterial coupling.

A 6-month-old Shiba Inu dog was brought to the Veterinary Medical Teaching Hospital because of a cough, exercise intolerance, and pulmonary edema. The dog had a Levine 2/6 systolic murmur. Transthoracic echocardiography revealed left atrial and ventricular dilatation (left atrium to aortic ratio: 2.8), mitral and tricuspid valve regurgitation, and severe left ventricular myocardial hypokinesia (fractional shortening was 11.8%). Bubble contrast echocardiography did not reveal a congenital shunt; therefore, the dog was clinically diagnosed with early onset dilated cardiomyopathy. From the first visit, the dog was treated with pimobendan, taurine, torasemide, and isosorbide dinitrate. After 435 days, echocardiography revealed that systolic function had not improved. On Day 465, atrial fibrillation was confirmed via electrocardiogram, and treatment with diltiazem hydrochloride was initiated. The dog continued to appear clinically stable thereafter, until it died suddenly 1087 days after the initial visit. A postmortem histopathological examination identified severe enlargement of the left atrial and ventricular chambers as well as attenuated wavy fibers in the ventricular myocardium, which confirmed dilated cardiomyopathy in a juvenile. This is the first report of a juvenile form of dilated cardiomyopathy in a Shiba Inu dog. This case report provides evidence that the extended prognosis of this dog differed from that in previously reported cases of dilated cardiomyopathy in young dogs. Key clinical message: This is the first reported case of a juvenile form of dilated cardiomyopathy in a Shiba Inu dog. This report provides evidence that the prognosis of this dog differed from that in previously reported cases of dilated cardiomyopathy in young dogs.

BACKGROUND: Hyperadrenocorticism (HAC) is a common endocrine disorder in dogs; however, there are no reports on the use of the corticotropin-releasing hormone test (CRHT) to differentiate between pituitary-dependent hyperadrenocorticism (PDH) and cortisol-producing adrenal tumors (CPATs), both causative of HAC. OBJECTIVES: To evaluate the usefulness of CRHT as a tool to differentiate between PDH and CPAT in dogs and to determine the reference intervals for CRHT in healthy, PDH, and CPAT dogs. ANIMALS: Dogs diagnosed with PDH (n = 21), CPAT (n = 6), and healthy beagle dogs (n = 33). METHODS: This prospective study included dogs with a definitive diagnosis of PDH and CPAT and healthy beagle dogs, in which CRHT was performed, were prospectively evaluated. We investigated the correlations of CRHT (endogenous adrenocorticotropic hormone [ACTH] concentration, endogenous ACTH concentration [EAC], and poststimulation ACTH concentration [PAC]) with pituitary-to-brain ratio (PBR) (in PDH) and with indices of adrenal ultrasonography (smaller and larger adrenal gland dorsoventral thickness in PDH and CPAT). RESULTS: For EAC, the area under the curve (AUC) was 0.95, with a cutoff value of 26.3 pg/mL (sensitivity: 90.62%, specificity: 87.50%). The AUC for PAC was 0.96 with a cutoff value of 54.5 pg/mL (sensitivity: 100.00%, specificity: 66.67%). The 95% reference interval for CRHT in healthy (control) dogs ranged 5.00 to 79.8 pg/mL (1.10-17.57 pmol/L) for EAC, and 1.92 to 153.42 pg/mL (0.42-33.78 pmol/L) for PAC. There was no significant correlation between PBR and CRHT, nor adrenal size and CRHT. CONCLUSIONS AND CLINICAL IMPORTANCE: CRHT appears to be a rapid and reliable test for differentiating PDH from CPAT in dogs.

Transsphenoidal surgery (TSS) is a curative treatment for pituitary-dependent hyperadrenocorticism, and its use in dogs has recently increased. One of the most serious postoperative complications of TSS is dyspnoea. We report three cases where transtracheal catheter oxygen therapy prevented death from respiratory distress secondary to enlarged soft palate after TSS.

Pulmonary hypertension (PH) is a life-threatening disease in dogs characterized by increased pulmonary arterial pressure (PAP) and/or pulmonary vascular resistance. No study has evaluated the utility of Beraprost sodium (BPS) in dogs with PH. This study aimed to evaluate the effect of BPS on cardiac function and hemodynamics and examine the optimal dose of BPS in canine models of chronic embolic PH. In this prospective crossover study, three doses of BPS (5, 15, and 25 μg/kg, twice a day) were examined in eight canine models of chronic embolic PH. All model dogs underwent invasive PAP measurement, echocardiography, and non-invasive systemic blood pressure measurement before and after continuous administration of oral BPS for 1 week. No side effects of BPS were observed in any dog during the study. All doses of BPS significantly decreased systolic PAP and pulmonary vascular impedance. Additionally, systemic vascular impedance significantly decreased with 15 and 25 μg/kg of BPS. The right ventricular stroke volume and longitudinal strain significantly decreased with all doses of BPS. The left ventricular stroke volume and circumferential strain decreased with 15 μg/kg BPS. BPS was well-tolerated in this study. A dose-dependent vasodilating effect on pulmonary vessels was observed in canine models of chronic PH. Additionally, 15 μg/kg BPS showed a balanced vasodilating effect on systemic and pulmonary vessels. Furthermore, with a decrease in systemic and pulmonary vascular impedance, the left and right ventricular functions were significantly improved. Our results suggest that BPS may be useful in the treatment of canine PH.

Pulmonary hypertension (PH) is a common comorbidity in dogs with myxomatous mitral valve disease (MMVD), and can induce various changes in the right heart, such as right ventricular (RV) hypertrophy, dilatation, and dysfunction. We hypothesized that RV function, not only systolic function but also diastolic function, could be worsened with PH progression. We aimed to compare RV systolic and diastolic function in dogs with MMVD. Twenty healthy dogs and sixty-eight dogs with MMVD were enrolled. Dogs with MMVD were classified into the probability of PH. Two-dimensional and Doppler echocardiographic indices for right heart and two-dimensional speckle tracking echocardiography indices were measured. The morphological indicators of the right heart were significantly higher only in the high probability of PH group. The RV strain, early-diastolic and systolic strain rates were significantly lower in the high probability of PH group than those in the low and intermediate probability of PH groups. Multivariate analysis showed that increased RV internal dimension normalized by body weight and RV myocardial performance index were significantly associated with the presence of right-sided congestive heart failure. Speckle tracking echocardiography-derived RV systolic and diastolic function were activated in the low and intermediate probability of PH groups. However, dogs with high probability of PH showed RV myocardial dysfunction and dilatation. Increased RV myocardial performance index and end-diastolic RV internal dimension normalized by body weight were significantly associated with the presence of right-sided congestive heart failure in dogs with MMVD.

OBJECTIVE: To evaluate the influence of manual ventilation-controlled respiration on right ventricular (RV) pressure-volume loop-derived and echocardiographic variables in dogs. ANIMALS: 8 healthy, anesthetized Beagles. PROCEDURES: In a prospective experimental study, pressure-volume catheters were percutaneously inserted into the right ventricle of each dog, and manual ventilation was performed; RV pressure-volume loop (hemodynamic) data and conventional echocardiographic variables were assessed. Two-dimensional speckle tracking echocardiography-derived RV strain (RVS) and RV systolic strain rate (RVSR) were obtained with RV free wall-only analysis (free wall) and RV global analysis (RVGA; interventricular septum). Variables were compared between end-inspiratory and end-expiratory phases of respiration by statistical methods. Multiple regression analysis was used to assess associations between selected hemodynamic and echocardiographic variables. RESULTS: The RV pressure significantly increased, and RV volume, stroke volume, tricuspid annular plane systolic excursion, RV fractional area change, peak myocardial systolic velocity of the lateral tricuspid annulus, and RV free wall only-assessed RVS and RVSR significantly decreased in the inspiratory phase, compared with the expiratory phase. There were no significant differences in end-systolic elastance or RVGA-assessed RVS or RVSR between respiratory phases. The RVGA-assessed RVSR was significantly associated with stroke volume and end-systolic elastance. CONCLUSIONS AND CLINICAL RELEVANCE: Specific RV echocardiographic variables were significantly affected by respiration. In contrast, RVS and RVSR determined with RVGA were not affected by respiration and were associated with hemodynamic indicators of RV contractility.

Post-capillary pulmonary hypertension (PH) is a life-threatening complication in dogs with myxomatous mitral valve disease (MMVD). An increase in pulmonary vascular resistance (PVR) is associated with post-capillary PH progression. In humans, PVR estimated by echocardiography (PVRecho) enables the non-invasive assessment of PVR in patients with PH. This study aimed to evaluate the clinical utility of PVRecho in dogs with MMVD, PH probability, and right-sided congestive heart failure (R-CHF). Dogs with MMVD and detectable tricuspid valve regurgitation were included in the study. Dogs were classified into three PH probability groups (low/intermediate/high) and according to the presence or absence of R-CHF. All dogs underwent echocardiographic measurements for right ventricular (RV) morphology and function. PVRecho was calculated by two methods using tricuspid valve regurgitation velocity and velocity–time integral of the pulmonary artery flow (PVRecho and PVRecho2). RV size indicators were significantly higher with a higher probability of PH. RV strain and velocity–time integral of the pulmonary artery flow in the high probability group were significantly lower than those in the other groups. Tricuspid valve regurgitation velocity, PVRecho, and PVRecho2 were significantly higher with an increase in PH probability. Logistic regression analysis revealed a significant association between the presence of R-CHF and increased PVRecho2 and end-diastolic RV internal dimension normalized by body weight. PVRecho and PVRecho2 showed significant differences among the PH probability groups. These non-invasive variables may be useful for the diagnosis and stratification of PH and the determination of the presence of R-CHF in dogs with MMVD.

BACKGROUND: Restrictive cardiomyopathy (RCM) is a common myocardial disease in cats, characterized by diastolic dysfunction and atrial enlargement without myocardial hypertrophy. Especially, endomyocardial form of RCM, one of the subtypes in RCM, is characterized by endocardial fibrosis, endocardial scar bridging the interventricular septum and left ventricular (LV) free wall, and deformation and distortion of the LV. However, it is unclear how the myocardial dysfunction and the endocardial scar contribute to the pathophysiology of RCM disease progression. CASE PRESENTATION: A 3 years and 2 months old, intact male, Domestic shorthaired cat was presented for consultation of cardiac murmur. At the first visit (day 0), the notable abnormal finding was echocardiography-derived chordae tendineae-like structure bridging the interventricular septum and the LV free wall, resulting high-speed blood flow in the left ventricle. Electrocardiography, thoracic radiography and noninvasive blood pressure measurements were normal. No left atrial enlargement was observed, and LV inflow velocity showed an abnormal relaxation pattern. Although there was no abnormality in tissue Doppler imaging-derived myocardial velocity, two-dimensional speckle tracking echocardiography (2D-STE) revealed a decrease in the LV longitudinal strain and an increase in endocardial to epicardial ratio of the LV circumferential strain on day 0. On day 468, obvious left atrium enlargement and smoke like echo in the left atrium were observed. The LV inflow velocity was fused, and the tissue Doppler imaging-derived early-diastolic myocardial velocity of the septal mitral annulus decreased. Regarding 2D-STE, LV circumferential strain was further decreased, and right ventricular strain was additionally decreased. Although the general condition was good, we made a clinical diagnosis of endomyocardial RCM based on the above findings. On day 503, the cat showed the radiographic evidence of pulmonary edema and congestive heart failure signs. CONCLUSIONS: Cats with abnormal LV structure and associated myocardial dysfunction like this case needs careful observation. Additionally, 2D-STE indices may be useful for early detection of myocardial dysfunction in feline RCM.

Adipose tissue-derived mesenchymal stem cells (ADSCs) have anti-inflammatory and immunomodulatory characteristics. Many studies have suggested that the immunomodulation of ADSCs is largely mediated by secreted paracrine factors. Various factors are secreted from ADSCs, among which extracellular vesicles are considered to play a major role in the communication between ADSCs and target cells. Several studies have reported the function of canine ADSC-derived extracellular vesicles (cADSC-EVs), but few studies have reported the immunomodulatory effects of cADSC-EVs on immune cells. The purpose of this study was to investigate the effects of cADSC-EVs on in vitro-stimulated CD4+ T cells isolated from peripheral blood mononuclear cells (PBMCs). cADSC-EVs were isolated from cADSCs under naive conditions or primed conditions by tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). The expression levels of several microRNAs in cADSC-EVs were altered by priming with TNFα and IFNγ. Culturing PBMCs stimulated with concanavalin A in the presence of naive or primed cADSC-EVs inhibited the differentiation of PBMCs and CD4+ T cells and promoted apoptosis of PBMCs. CD4+, CD8+, and CD4+CD8+ T cells were decreased, while CD3+CD4-CD8- T cells were increased. T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cells were analyzed by flow cytometry. cADSC-EVs inhibited the proliferation of Th1 and Th17 cells and enhanced Th2 and Treg cell proliferation. However, CD4+ T cells that had incorporated labeled cADSC-EVs comprised only a few percent of all cells. Therefore, these responses of stimulated CD4+ T cells may be due to not only direct effects of cADSC-EVs but also to indirect effects through interactions between cADSC-EVs and other immune cells. In conclusion, cADSC-EVs exert immunosuppressive effects on stimulated CD4+ T cells in vitro. These findings may be useful for further studies of immune diseases.

The endomyocardial form of restrictive cardiomyopathy (EMF-RCM), a primary disorder of the myocardium, is one of the diseases with poor prognosis in cats. We hypothesized that both the left and right myocardial functional abnormalities may occur in cats with EMF-RCM, causing this disease pathophysiology and clinical status. Out of the 25 animals included in this study, 10 were client-owned cats with EMF-RCM, and 15 were healthy cats. In this study, cats were assessed for layer-specific myocardial function (whole, endocardial, and epicardial) in the left ventricular longitudinal and circumferential directions, and right ventricular longitudinal direction, via two-dimensional speckle-tracking echocardiography (2D-STE). Cats with EMF-RCM had depressed left ventricular myocardial deformations both in systole (whole longitudinal strain, epicardial longitudinal strain, and endocardial circumferential strain) and diastole (early and late diastolic longitudinal strain rates, and late diastolic circumferential strain rate) compared to controls. Furthermore, some right ventricular myocardial deformations (systolic longitudinal strain in epicardial layers, and endocardial-to-epicardial strain ratio) were significantly differerent in cats with EMF-RCM. Myocardial function assessed by 2D-STE could reveal left and right myocardial dysfunction.

Congestive heart failure (CHF) is a life-threatening condition in cats with cardiomyopathy. We hypothesized that myocardial dysfunction may induce progression to CHF pathophysiology in cats with cardiomyopathy. However, no previous studies have evaluated the involvement of myocardial dysfunction in cats with CHF. In this study, we aimed to evaluate the relationship between CHF and myocardial function assessed using two-dimensional speckle-tracking echocardiography (2D-STE). Sixteen client-owned healthy cats and 32 cats with cardiomyopathy were enrolled in this study. Cats were classified into three groups: healthy cats, cardiomyopathy without CHF (CM group), and cardiomyopathy with CHF (CHF group). Left ventricular (LV) longitudinal and circumferential strains (SL and SC, respectively), and right ventricular (RV) SL were measured using 2D-STE. Logistic regression analysis was performed to assess the relationship between CHF and echocardiographic variables, including 2D-STE. Results comparing the healthy cats and CM vs. CHF groups showed that increased left atrial to aortic diameter ratio and decreased LV apical SC were significantly associated with the existence of CHF (odds ratio [95% confidence interval]: 1.40 [1.16-1.78] and 1.59 [1.06-2.36], respectively). Results comparing the CM vs. CHF group showed that increased end-diastolic RV internal dimension and decreased RV SL were significantly associated with the existence of CHF (odds ratio: 1.07 [1.00-1.13] and 1.34 [1.07-1.68], respectively). Left atrial enlargement and depressed LV apical myocardial function may be useful tools for predicting the progression to CHF in cats. Furthermore, RV enlargement and dysfunction may lead to the onset of CHF in asymptomatic cats with cardiomyopathy.

BACKGROUND: Tricuspid annular plane systolic excursion (TAPSE) is a common right ventricular (RV) function indicator. However, TAPSE was not decreased in dogs with myxomatous mitral valve disease (MMVD) and postcapillary pulmonary hypertension (PH) because of its load, angle, and body weight dependency, and TAPSE was considered a preload-dependent index. OBJECTIVES: To evaluate the utility of TAPSE normalized by RV size in dogs with postcapillary PH. ANIMALS: Twenty healthy dogs and 71 MMVD dogs with or without PH. METHODS: In this prospective observational study, end-diastolic RV internal dimension (RVIDd), end-diastolic and end-systolic RV area, and end-diastolic RV wall thickness were measured as RV size indices. The TAPSE was measured using B-mode and M-mode methods. Normalized TAPSE was calculated by dividing TAPSE by each RV size index. The RV strain was obtained as the detailed RV function using 2-dimensional speckle tracking echocardiography. All indices were compared among the PH severity groups and in the presence of right-sided congestive heart failure (R-CHF). RESULTS: Although nonnormalized TAPSE was higher with PH severity progression, each normalized TAPSE showed a significant decrease in the severe PH group (P < .05). The TAPSEB-mode /RVIDd ratio had high area under the curve to predict R-CHF and had moderate correlation with RV strain (P < .05). The TAPSEB-mode /RVIDd and left atrial-to-aortic diameter ratios were independent predictors for R-CHF. CONCLUSIONS AND CLINICAL IMPORTANCE: Normalized TAPSE could reflect RV systolic dysfunction in dogs with severe PH, which could not be detected by nonnormalized TAPSE. The TAPSEB-mode /RVIDd ratio might predict R-CHF with high sensitivity and reproducibility.

In human medicine, fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridioides difficile infection. It has also been tested as a treatment for multiple gastrointestinal diseases, including inflammatory bowel disease (IBD). However, only a few studies have focused on the changes in the microbiome following FMT for canine IBD. Here, we performed FMT in nine dogs with IBD using the fecal matter of healthy dogs and investigated the subsequent changes in the fecal microbiome and clinical signs. In three dogs, the fecal microbiome was examined by 16S rRNA sequencing. Fusobacteria were observed at a low proportion in dogs with IBD. However, the post-FMT microbiome became diverse and showed a significant increase in Fusobacteria proportion. Fusobacterium was detected in the nine dogs by quantitative polymerase chain reaction. The proportion of Fusobacterium in the post-FMT fecal microbiome was significantly increased (p<0.05). The changes in clinical signs (e.g., vomiting, diarrhea, and weight loss) were evaluated according to the canine inflammatory bowel disease activity index. The score of this index significantly decreased in all dogs (p<0.05) with improvements in clinical signs. These improvements were related to the changes in the proportion of microbes, particularly the increase in Fusobacterium. The dogs with IBD showed a lower proportion of Fusobacterium than healthy dogs. This suggests that a low proportion of Fusobacterium is a characteristic feature of canine IBD and that Fusobacterium is involved in this disease. The results of this study may help elucidate the pathogenesis of this disease and its association with Fusobacterium.

Background: Pulmonary hypertension (PH) is a life-threatening disease in dogs characterized by an increase in pulmonary arterial pressure (PAP) and/or pulmonary vascular resistance. Right ventricle adapts to its pressure overload through various right ventricular (RV) compensative mechanisms: adaptive and maladaptive remodeling. The former is characterized by concentric hypertrophy and increased compensatory myocardial contractility, whereas the latter is distinguished by eccentric hypertrophy associated with impaired myocardial function. Objectives: To evaluate the RV adaptation associated with the increase of PAP using two-dimensional speckle tracking echocardiography. Animals: Seven experimentally induced PH models. Methods: Dogs were anesthetized and then a pulmonary artery catheter was placed via the right jugular vein. Canine models of PH were induced by the repeated injection of microspheres through the catheter and monitored pulmonary artery pressure. Dogs were performed echocardiography and hemodynamic measurements in a conscious state when baseline and systolic PAP (sPAP) rose to 30, 40, 50 mmHg, and chronic phase. The chronic phase was defined that the sPAP was maintained at 50 mmHg or more for 4 weeks without injection of microspheres. Results: Pulmonary artery to aortic diameter ratio, RV area, end-diastolic RV wall thickness, and RV myocardial performance index were significantly increased in the chronic phase compared with that in the baseline. Tricuspid annular plane systolic excursion was significantly decreased in the chronic phase compared with that in the baseline. The RV longitudinal strain was significantly decreased in the sPAP30 phase, increased in the sPAP40 and sPAP50 phases, and decreased in the chronic phase. Conclusions: Changes in two-dimensional speckle tracking echocardiography-derived RV longitudinal strain might reflect the intrinsic RV myocardial contractility during the PH progression, which could not be detected by conventional echocardiographic parameters.

The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide μ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.

Purpose: Recently, fecal microbiota transplantation (FMT) has been tested in veterinary medicine as a treatment option for multiple gastrointestinal (GI) diseases, such as inflammatory bowel disease (IBD). However, there are no reports of changes in the microbial diversity of fecal microbiome after treatment with FMT in canine IBD cases. Moreover, little is known about the long-term efficacy and safety of FMT treatment for dogs. Herein, we present a case of canine intractable IBD treated with repeated, long-term FMT. Patients and methods: The patient was a 10-year-old, neutered, male, 4-kg Toy Poodle with a prolonged history of vomiting and diarrhea. Fecal examination for pathogens was negative. Despite treatment with multiple antibacterial and antidiarrheal agents, the patient showed no improvement. Endoscopic mucus sampling diagnosed a case of lymphocytic-plasmacytic duodenitis, ie, idiopathic IBD. Eventually, we performed periodic, long-term fecal microbiota transplantation of fresh donor feces collected from a 4-year-old, 32.8-kg, neutered male Golden Retriever by rectal enema. Additionally, we performed 16S rRNA sequence analysis, before and after FMT, to evaluate the microbiome diversity. Results: Fecal microbiome diversity after FMT resembled that of the healthy donor dog's fecal microbiome, before FMT, which led us to conclude that the fecal microbiome in our patient normalized with FMT. Moreover, the clinical symptoms improved remarkably with regard to the changes in the fecal microbiome. Additionally, we noted no observable side effects during FMT treatment. Conclusion: This report indicates the efficacy and safety of long-term, periodic FMT for a case of canine IBD based on attenuation of clinical symptoms and changes in fecal microbiome diversity. Therefore, FMT could be chosen as a treatment option for IBD in canines in the future.

Adipose-derived mesenchymal stem cells (ADSCs) have been suggested their benefits in regenerative medicine for various diseases. Lipomas, benign neoplasms in adipose tissue, have been reported as a potential source of stem cells. These lipoma-derived mesenchymal stem cells (LDSCs) may be useful for regenerative medicine. However, the detailed characteristics of LDSCs have not been fully elucidated. This study investigated the cellular proteomics and secretomes of canine LDSCs in addition to morphology and proliferation and differentiation capacities. Some LDSCs isolated from canine subcutaneous lipomas were morphologically different from ADSCs and showed a rounded shape instead of fibroblast-like morphology. The phenotype of cell surface markers in LDSCs was similar to those in ADSCs, but CD29 and CD90 stem cell markers were more highly expressed compared with those of ADSCs. LDSCs had noticeably high proliferation ability, but no significant differences were observed compared with ADSCs. In regard to differentiation capacity compared to ADSCs, LDSCs showed higher adipogenesis, but no differences were observed with osteogenesis. Cellular proteomic analysis using two-dimensional gel electrophoresis revealed that over 95% of protein spots showed similar expression levels between LDSCs and ADSCs. Secretome analysis was performed using iTRAQ and quantitative cytokine arrays. Over 1900 proteins were detected in conditioned medium (CM) of LDSCs and ADSCs, and 94.0% of detected proteins showed similar expression levels between CM of both cell types. Results from cytokine arrays including 20 cytokines showed no significant differences between CM of LDSCs and that of ADSCs. Our results indicate that canine LDSCs had variability in characteristics among individuals in contrast with those of ADSCs. Cellular proteomics and secretomes were similar in both LDSCs and ADSCs. These findings suggest that LDSCs may be suitable for application in regenerative medicine.

The potential effects of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the growth and invasion of canine tumours including hepatocellular carcinoma (HCC) are not yet understood. Moreover in humans, the functional contribution of AT-MSCs to malignancies remains controversial. The purpose of this study was to investigate the effects of AT-MSCs on the proliferation and invasion of canine HCC cells in vitro. The effect of AT-MSCs on mRNA levels of factors related to HCC progression were also evaluated. Conditioned medium from AT-MSCs (AT-MSC-CM) significantly enhanced canine HCC cell proliferation and invasion. Moreover, mRNA expression levels of transforming growth factor-beta 1, epidermal growth factor A, hepatocyte growth factor, platelet-derived growth factor-beta, vascular endothelial growth factor, and insulin-like growth factor 2 were 2.3 ± 0.4, 2.0 ± 0.5, 5.7 ± 1.9, 1.7 ± 0.2, 2.1 ± 0.4, and 1.4 ± 0.3 times higher, respectively (P &lt 0.05). The mRNA expression level of MMP-2 also increased (to 4.0 ± 1.2 times control levels) in canine HCC cells co-cultured with AT-MSCs, but MMP-9 mRNA significantly decreased (to 0.5 ± 0.1 times control levels). These findings suggest that soluble factors from AT-MSCs promote the proliferation and invasion of canine HCC cells.

Objectives Hypertrophic cardiomyopathy, a primary disorder of the myocardium, is the most common cardiac disease in cats. However, determination of myocardial deformation with two-dimensional speckle-tracking echocardiography in cats with various stages of hypertrophic cardiomyopathy has not yet been reported. This study was designed to measure quantitatively multidirectional myocardial deformations of cats with hypertrophic cardiomyopathy. Methods Thirty-two client-owned cats with hypertrophic cardiomyopathy and 14 healthy cats serving as controls were enrolled and underwent assessment of myocardial deformation (peak systolic strain and strain rate) in the longitudinal, radial and circumferential directions. Results Longitudinal and radial deformations were reduced in cats with hypertrophic cardiomyopathy, despite normal systolic function determined by conventional echocardiography. Cats with severely symptomatic hypertrophic cardiomyopathy also had lower peak systolic circumferential strain, in addition to longitudinal and radial strain. Conclusions and relevance Longitudinal and radial deformation may be helpful in the diagnosis of hypertrophic cardiomyopathy. Additionally, the lower circumferential deformation in cats with severe hypertrophic cardiomyopathy may contribute to clinical findings of decompensation, and seems to be related to severe cardiac clinical signs. Indices of multidirectional myocardial deformations by two-dimensional speckle-tracking echocardiography may be useful markers and help to distinguish between cats with hypertrophic cardiomyopathy and healthy cats. Additionally, they may provide more detailed assessment of contractile function in cats with hypertrophic cardiomyopathy.

Introduction: Determine the usefulness of dyssynchrony indices derived from two-dimensional speckle tracking echocardiography for the detection of mechanical dyssynchrony in a canine model of left bundle branch block. Animals: Ten healthy beagles. Materials and Methods: Segmental, time-radial strain curves were obtained using two-dimensional speckle tracking echocardiography. The maximum difference and standard deviation of the time to peak radial strain for six predefined segments (MaxD-TpSR and 6SD-TpSR) were calculated, together with the left ventricular dyssynchrony by radial strain (DysSR), before and after ablation of the left bundle branch block. Receiver operating characteristic curve analysis was performed using dogs after ablation as positive controls. Results: After ablation, all dogs showed multiple peaks in at least one segment on the time radial strain curve, while all dyssynchrony indices increased significantly (MaxD-TpSR from 16.25 +/- 16.04 [mean standard deviation] to 44.4 +/- 26.18 ms, 6SD-TpSR from 7.59 +/- 7.40 to 19.62 +/- 11.91 ms, and DysSR from 4.20 +/- 2.12 to 10.87 +/- 2.92%, p&lt;0.05). In receiver operating characteristic curve analysis, the areas under the curve for MaxD-TpSR, 6SD-TpSR, and DysSR were 0.825, 0.800, and 0.980, respectively. Conclusions: Left ventricular dyssynchrony by radial strain can detect mechanical dyssynchrony with higher sensitivity and specificity than dyssynchrony indices, based on the time to peak radial strain. (C) 2016 Elsevier B.V. All rights reserved.