Surgical Removal of a Canine Aortic Thromboembolism Secondary to Pancreatitis

A 7-year-old castrated male Pomeranian was evaluated on emergency for diagnostic work-up and treatment for acute nonpainful paraparesis. The neurologic examination suggested a L4-S3 myelopathy, but physical examination revealed lack of femoral pulses and rectal hypothermia, as well as a grade II/VI systolic heart murmur, so ischemic neuromyopathy was suspected. Clinicopathologic analysis revealed increased muscle enzymes and proteinuria. Abdominal ultrasonography confirmed aortic thromboembolism (ATE), and surgical histopathology diagnosed necrotizing pancreatitis. Surgical aortic thrombectomy was performed, and antithrombotic therapy was instituted. Pancreatitis was treated supportively. The dog was discharged to the owners after 10 days of hospitalization. Recheck examination 6 weeks after initial presentation revealed a normal neurologic examination and normal femoral pulses. The patient has had no further bouts of pancreatitis and remains neurologically normal 5 years after initial presentation. Canine ATE is relatively rare compared to the feline counterpart. Directed therapy for feline ATE is often not recommended, as underlying conditions are oftentimes ultimately fatal. Underlying etiologies for canine ATE include cardiovascular disease and endocrinopathies, but canine ATE secondary to pancreatitis has not yet been reported. Surgical removal of aortic thromboembolus should be considered as curative for pelvic limb dysfunction in the canine patient without a terminal underlying disease

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration–approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 1013 vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy

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

Abstract 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

Mast Cell Stabilization Decreases Cardiomyocyte and LV Function in Dogs with Isolated Mitral Regurgitation.

BACKGROUND: Mast cells are increased in isolated mitral regurgitation (MR) in the dog and may mediate extracellular matrix loss and left ventricular (LV) dilatation. We tested the hypothesis that mast cell stabilization would attenuate LV remodeling and improve function in the MR dog. METHODS AND RESULTS: MR was induced in adult dogs randomized to no treatment (MR, n = 5) or to the mast cell stabilizer, ketotifen (MR + MCS, n = 4) for 4 months. LV hemodynamics were obtained at baseline and after 4 months of MR and magnetic resonance imaging (MRI) was performed at sacrifice. MRI-derived, serial, short-axis LV end-diastolic (ED) and end-systolic (ES) volumes, LVED volume/mass ratio, and LV 3-dimensional radius/wall thickness were increased in MR and MR + MCS dogs compared with normal dogs (n = 6) (P \u3c .05). Interstitial collagen was decreased by 30% in both MR and MR + MCS versus normal dogs (P \u3c .05). LV contractility by LV maximum time-varying elastance was significantly depressed in MR and MR + MCS dogs. Furthermore, cardiomyocyte fractional shortening was decreased in MR versus normal dogs and further depressed in MR + MCS dogs (P \u3c .05). In vitro administration of ketotifen to normal cardiomyocytes also significantly decreased fractional shortening and calcium transients. CONCLUSIONS: Chronic mast cell stabilization did not attenuate eccentric LV remodeling or collagen loss in MR. However, MCS therapy had a detrimental effect on LV function because of a direct negative inotropic effect on cardiomyocyte function

Chronic β1-adrenergic blockade enhances myocardial β3-adrenergic coupling with nitric oxide-cGMP signaling in a canine model of chronic volume overload: new insight into mechanisms of cardiac benefit with selective β1-blocker therapy.

The β1-adrenergic antagonist metoprolol improves cardiac function in animals and patients with chronic heart failure, isolated mitral regurgitation (MR), and ischemic heart disease, though the molecular mechanisms remain incompletely understood. Metoprolol has been reported to upregulate cardiac expression of β3-adrenergic receptors (β3AR) in animal models. Myocardial β3AR signaling via neuronal nitric oxide synthase (nNOS) activation has recently emerged as a cardioprotective pathway. We tested whether chronic β1-adrenergic blockade with metoprolol enhances myocardial β3AR coupling with nitric oxide-stimulated cyclic guanosine monophosphate (β3AR/NO-cGMP) signaling in the MR-induced, volume-overloaded heart. We compared the expression, distribution, and inducible activation of β3AR/NO-cGMP signaling proteins within myocardial membrane microdomains in dogs (canines) with surgically induced MR, those also treated with metoprolol succinate (MR+βB), and unoperated controls. β3AR mRNA transcripts, normalized to housekeeping gene RPLP1, increased 4.4 × 10(3)- and 3.2 × 10(2)-fold in MR and MR+βB hearts, respectively, compared to Control. Cardiac β3AR expression was increased 1.4- and nearly twofold in MR and MR+βB, respectively, compared to Control. β3AR was detected within caveolae-enriched lipid rafts (Cav3(+)LR) and heavy density, non-lipid raft membrane (NLR) across all groups. However, in vitro selective β3AR stimulation with BRL37344 (BRL) triggered cGMP production within only NLR of MR+βB. BRL induced Ser (1412) phosphorylation of nNOS within NLR of MR+βB, but not Control or MR, consistent with detection of NLR-specific β3AR/NO-cGMP coupling. Treatment with metoprolol prevented MR-associated oxidation of NO biosensor soluble guanylyl cyclase (sGC) within NLR. Metoprolol therapy also prevented MR-induced relocalization of sGCβ1 subunit away from caveolae, suggesting preserved NO-sGC-cGMP signaling, albeit without coupling to β3AR, within MR+βB caveolae. Chronic β1-blockade is associated with myocardial β3AR/NO-cGMP coupling in a microdomain-specific fashion. Our canine study suggests that microdomain-targeted enhancement of myocardial β3AR/NO-cGMP signaling may explain, in part, β1-adrenergic antagonist-mediated preservation of cardiac function in the volume-overloaded heart

Chymase Inhibition Prevents Fibronectin and Myofibrillar Loss and Improves Cardiomyocyte Function and LV Torsion Angle in Dogs with Isolated Mitral Regurgitation.

BACKGROUND: The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. METHODS AND RESULTS: We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a \u3e40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P CONCLUSIONS: These results suggest that chymase disrupts cell surface-fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)–mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification–mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart’s valves. S. aureus relies on virulence factors to evade therapeutics and the host’s immune response, usurping the host’s clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor–binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor–binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart’s valves. S. aureus relies on virulence factors to evade therapeutics and the host’s immune response, usurping the host’s clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor–binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor–binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases