Leslie C. Sharkey

Adipokines: a review of biological and analytical principles and an update in dogs, cats, and horses

In addition to its role as an energy storage depot, adipose tissue is now recognized as a complex endocrine organ. Adipose tissue releases a variety of factors, termed adipokines, that regulate energy metabolism, cardiovascular function, reproductive status, and immune function. Some of the better-studied adipokines include leptin, adiponectin, and components of the renin-angiotensin system such as angiotensinogen. The function of more recently discovered adipokines such as resistin are under intense scrutiny. Abnormal production or regulation of adipokines occurs in obese individuals and is implicated in the development of a variety of associated co-morbidities including metabolic syndrome, type 2 diabetes, atherosclerosis, heart disease, and cancer in people, although evaluation in domestic species is just beginning. Adipokines are now being examined as potential biomarkers for risk assessment for development of complications related to obesity. This article summarizes the function and regulation of some better-characterized adipokines. It also reviews the current information on the characterization of adipokines in some domestic species in which rates of obesity and obesity-related disorders are increasing, such as the dog, cat, and horse.

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma

BackgroundThe etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease.MethodsWe first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR.ResultsEach of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma).ConclusionsThe data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.

Evaluation of serum cardiac troponin I concentration in dogs with renal failure

OBJECTIVE To determine whether dogs with renal failure have higher serum cardiac troponin I (cTnI) concentrations than healthy dogs. DESIGN Case-control study. ANIMALS 31 dogs with renal failure and 51 healthy dogs. PROCEDURES Serum concentrations of creatinine and cardiac troponin I, urine specific gravity, and systolic arterial blood pressure were measured for all dogs. Dogs underwent a standardized physical examination, and any dog with evidence of cardiovascular disease or other nonrenal disease was excluded from final analyses. Dogs were considered to be in renal failure when the serum creatinine concentration was >or= 3.0 mg/dL, urine specific gravity was between 1.007 and 1.030, and renal failure had been clinically diagnosed. RESULTS Dogs with renal failure had significantly higher serum cTnI concentrations (median, 0.35 ng/mL) than did healthy dogs (0.20 ng/mL). The renal failure group also had a significantly higher median systolic blood pressure (156 mm Hg) than did healthy dogs (138 mm Hg), although serum cTnI concentration was not correlated with systolic blood pressure in dogs with renal failure. There was no significant difference in age between dogs with renal failure and healthy dogs, but dogs with renal failure had significantly higher serum creatinine concentration and lower urine specific gravity. CONCLUSIONS AND CLINICAL RELEVANCE Although dogs with renal failure did not have overt clinical signs of cardiac disease, they had high serum cTnI concentrations, which may have been associated with subclinical cardiovascular disease. The cause of the high serum cTnI concentration in these dogs requires additional investigation.

Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas.

Spontaneous pregnancy-induced hypertension and intrauterine growth restriction in rats

The SHHF/Mcc-fa(cp) (spontaneous hypertension and heart failure) rat is advanced as a novel and suitable non-primate model of pregnancy-associated hypertension and fetal growth restriction because it simultaneously has spontaneous pregnancy-associated hypertension, small for gestational age (SGA) offsprings, and altered placental gene expression. Pregnancy-associated hypertension is a major contributor to maternal and fetal morbidity and mortality with the potential to result in maternal death and the need for iatrogenic preterm delivery. It has been reported to develop spontaneously in humans, but not in animals; consequently, progress in identifying the cause and pathogenesis of this disorder has been hampered. Spontaneous hypertension and heart failure rats develop hypertension spontaneously as they age, therefore we sought to determine whether these rats developed hypertension and SGA offsprings during pregnancy. Our results show that systolic blood pressure (BP) increased >40 mm Hg by the end of the first trimester and remained at this elevated level for the remainder of pregnancy, but decreased after parturition. Placenta weights of SHHF rats (0.60 +/- 0.02 g, n = 36) were significantly higher than Wistar-Kyoto (WKY) rats (0.42 +/- 0.01 g, n = 22, P < .05), but pup weights were significantly lower (2.68 +/- 0.06 g for SHHF rats compared to 3.24 +/- 0.06 g for WKY controls, P < .05). Histologic examination revealed pathologic lesions in neither heart, liver, placenta, nor kidney. L-Arginine administered in drinking water prevented the elevation of BP, particularly during the third trimester. Placentas from SHHF rats displayed altered expression of several genes whose protein products have been implicated in preeclampsia, including serotonin receptor, sodium channel, carbonic anhydrase, estrogen receptor regulator, major histocompatibility complex proteins, superoxide dismutase, and angiotensiogen. In addition, gene expression profiling showed alteration of a number of subcellular putative myristoylproteins not previously associated with preeclampsia, particularly those engaged in post-translational modifications in the placenta. Thus, SHHF rats may be a valuable tool, because it simultaneously has spontaneous pregnancy-associated hypertension, SGA offsprings, and altered placental gene expression.

Correlation between cytologic and histopathologic diagnoses of bone lesions in dogs: a study of the diagnostic accuracy of bone cytology.

BACKGROUND The diagnostic value of cytology compared with histopathology varies by tissue, but there is little information regarding this comparison involving canine bone. OBJECTIVES The objective of this retrospective study was to compare primary pathologic processes for cytology and histopathology of canine bone lesions. We adopted a proposed standardized format for reporting studies of diagnostic accuracy. METHODS A computer search of canine medical records at the University of Minnesota Veterinary Medical Center from September 2002 through October 2006 identified 52 bone cytology samples that had incisional (IncB) and/or excisional (ExcB) biopsy performed. The primary pathologic process was determined by evaluation of original reports. Cytologic vs IncB and cytologic vs ExcB were compared pairwise for agreement. Agreement was compared for neoplastic and non-neoplastic processes using the combined IncB/ExcB data, which included all ExcB (n=21) and IncB when that was the only biopsy available (n=31). Combined data were used to determine the effect of cytology cellularity on the diagnostic correlation. RESULTS The correlation in primary process between cytology and IncB was 71%, and for ExcB was 71%. For lesions with a cytologic diagnosis of neoplasia compared with the combined IncB/ExcB data set, cytology and histopathology agreed in 92% of cases, which was significantly greater (P<.0001, chi2) than the 27% for non-neoplastic processes. Cytology cellularity significantly affected rates of correlation (P=.026), with high, moderate, and poor cellularity samples having concordant primary processes in 88%, 77%, and 47% of cases, respectively. CONCLUSIONS Cytologic diagnosis of neoplasia for samples collected from canine bone correlates better with histopathology than cytologic diagnosis of non-neoplastic proliferative processes or inflammation. Cytologic diagnoses from highly cellular samples are more likely to correlate with histopathology than those from less cellular samples.

Utility of diagnostic tests for and medical treatment of pulmonary blastomycosis in dogs: 125 cases (1989-2006)

OBJECTIVE To compare results of the most common diagnostic tests for pulmonary blastomycosis in dogs, identify factors associated with outcome, and determine response to various antifungal treatment protocols. DESIGN Retrospective case series. ANIMALS 125 dogs with pulmonary blastomycosis. PROCEDURES Medical records were reviewed, and information was obtained regarding diagnostic methods, results of routine laboratory testing, and radiographic response to antifungal treatment. RESULTS 79 dogs survived, 38 died, and 8 were euthanized. Transthoracic fine-needle aspiration and transtracheal lavage were the most common diagnostic methods. Results of an agar gel immunodiffusion test for antibodies against Blastomyces dermatitidis were negative in 12 of 24 (50%) dogs. Only 3 of 94 (3.2%) dogs in which cytologic or histologic examination or bacterial culture of pulmonary samples were performed had any evidence of concurrent bacterial infection. The half-time for radiographic resolution of pulmonary infiltrates did not vary significantly with antifungal treatment, and use of a loading dosage of itraconazole was not associated with significant improvements in outcome or time to disease resolution. Dogs that died had a higher number of band neutrophils at initial examination, compared with those that survived. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the agar gel immunodiffusion test should not be used as the sole diagnostic test for pulmonary blastomycosis in dogs, that concurrent bacterial pneumonia was uncommon in dogs with pulmonary blastomycosis, and that the rate with which pulmonary infiltrates resolved did not vary significantly among antifungal treatments.

Effect of Pancreatic Tissue Sampling on Serum Pancreatic Enzyme Levels in Clinically Healthy Dogs

Little is known about the potential consequences of pancreatic tissue sampling in dogs. The goal of the present study was to evaluate changes in serum trypsin–like immunoreactivity and canine-specific pancreatic lipase after pancreatic fine-needle aspiration and surgical biopsy in 27 clinically healthy dogs. Presurgical, ultrasound-guided aspiration of the pancreas was performed with the dogs under sedation. Subsequently, all the dogs underwent intraoperative pancreatic fine-needle aspiration and clamshell biopsy. After euthanasia, pancreata were sectioned for histopathologic evaluation. Serum pancreatic enzyme levels were measured at 3 time points: baseline, after ultrasound-guided aspiration, and after intraoperative aspiration and biopsy. No significant differences were detected among mean serum pancreatic lipase values at any point (P > 0.05). Serum trypsin–like immunoreactivity did not change from baseline (18.2 ± 2.1 μg/dl; mean ± standard error) after ultrasound-guided aspiration (13.6 ± 2.2 μg/dl) but increased significantly after intraoperative sampling (44.8 ± 1.9 μg/dl; P < 0.0005). After surgical biopsy, the 20 dogs that had both ultrasound-guided and intraoperative sampling had a higher mean (SE) serum trypsin–like immunoreactivity (44.8 ± 1.9 μg/dl) than the 7 dogs that had only intraoperative samples taken (36.4 ± 4.1 μg/dl; P < 0.05). All 27 pancreata were grossly normal before intraoperative sampling. Pancreatic sampling was associated with increased serum trypsin–like immunoreactivity and mild, peracute necrosis, inflammation, hemorrhage, and fibrin deposition. Tissue damage from sampling was not sufficient to cause an elevation in canine-specific pancreatic lipase in the time frame evaluated. Further studies are needed to determine longer-term effects of pancreatic sampling on enzyme levels and clinical outcome.

Effect of ovariectomy in heart failure-prone SHHF/Mcc-facp rats

The importance of the loss of ovarian function to the progression of hypertension and heart disease in women is controversial. We investigated whether ovariectomy would accelerate development of hypertension, congestive heart failure, and neurohumoral activation in adult spontaneous hypertension heart failure (SHHF) rats, a genetic model of heart failure. Six months after ovariectomy, no significant differences between control and ovariectomized rats were seen in systolic or diastolic blood pressure, left ventricular fractional shortening by echocardiography, or heart weight. Percent V1 myosin isozyme was significantly lower in ovariectomized rats. Northern blot analysis failed to show significant differences between groups in expression of hepatic angiotensinogen, renal renin, or left ventricular atrial or brain natriuretic peptide mRNA. In a second experiment, serial measures of systolic pressure and left ventricular shortening fractions failed to document a significant difference between control and ovariectomized rats as they developed heart failure, although there was a significant decline in shortening fraction in both groups at the age when regular estrous cycling naturally ceases. Survival time was similar between groups. In summary, ovariectomy of adult SHHF rats does not appear to affect the progression of genetically programmed hypertension and heart failure in this model.The importance of the loss of ovarian function to the progression of hypertension and heart disease in women is controversial. We investigated whether ovariectomy would accelerate development of hypertension, congestive heart failure, and neurohumoral activation in adult spontaneous hypertension heart failure (SHHF) rats, a genetic model of heart failure. Six months after ovariectomy, no significant differences between control and ovariectomized rats were seen in systolic or diastolic blood pressure, left ventricular fractional shortening by echocardiography, or heart weight. Percent V1 myosin isozyme was significantly lower in ovariectomized rats. Northern blot analysis failed to show significant differences between groups in expression of hepatic angiotensinogen, renal renin, or left ventricular atrial or brain natriuretic peptide mRNA. In a second experiment, serial measures of systolic pressure and left ventricular shortening fractions failed to document a significant difference between control and ovariectomized rats as they developed heart failure, although there was a significant decline in shortening fraction in both groups at the age when regular estrous cycling naturally ceases. Survival time was similar between groups. In summary, ovariectomy of adult SHHF rats does not appear to affect the progression of genetically programmed hypertension and heart failure in this model.

Accuracy of US-guided FNA of focal liver lesions in dogs: 140 cases (2005-2008)

Medical records from dogs having abdominal ultrasound (US) performed between March 2005 and October 2008 were reviewed for detection of focal liver lesions (FLL) with both cytologic and histologic sampling. Samples were classified as to either the presence or absence of major categories of pathologic processes, including malignant neoplasia, inflammation, hyperplasia/benign neoplasia, vacuolar change, extramedullary hematopoeisis, cholestasis, necrosis, and no microscopic abnormalities. Evaluation of selection bias was performed by review of the relative distribution of cytologic diagnoses for cases with histology compared with cases excluded from the comparison analysis because histology results were not available. Cytology had the highest sensitivity for vacuolar change (57.9%), followed by neoplasia (52.0%). Cytology had the highest positive predictive value (PPV) for neoplasia (86.7%) followed by vacuolar change (51.6%). Cytology had lower sensitivity and PPVs for inflammation, necrosis, and hyperplasia. The ability of cytology to characterize disease in canine FLL varies by pathologic process. Clinicians can have a high degree of confidence when a cytologic diagnosis of neoplasia is given; however, cytology is less reliable for excluding the potential for neoplasia. Cytology has a low sensitivity and PPV for inflammation and a limited diagnostic performance for the diagnosis of vacuolar change.