Temporal changes in myocardial collagen, matrix metalloproteinases, and their tissue inhibitors in the left ventricular myocardium in experimental chronic mitral regurgitation in rodents.

Mitral regurgitation (MR) imposes left ventricular volume overload, triggering rapid ventricular dilatation, increased myocardial compliance, and, ultimately, cardiac dysfunction. Breakdown of the extracellular matrix has been hypothesized to drive these rapid changes, partially from an imbalance in the matrix metalloproteinases (MMPs) and their tissue inhibitors [tissue inhibitors of metalloproteinase (TIMPs)]. In the present study, we developed a rat model of severe MR that mimics the human condition and investigated the temporal changes in extracellular matrix-related genes, collagen biosynthesis proteins, and proteolytic enzymes over a 20-wk period. Male Sprague-Dawley rats were anesthetized to a surgical plane with mechanical ventilation, and a thoracotomy was performed to expose the apex. Using transesophageal ultrasound guidance, a needle was inserted into the beating heart to perforate the anterior mitral leaflet and create severe MR. Animals were survived for 20 wk, with some animals terminated at 2, 10, and 20 wk for analysis of left ventricular tissue. A sham group that underwent the same surgery without mitral leaflet perforation and MR were used as controls. At 2 wk post-MR, increased collagen gene expression was measured, but protein levels of collagen did not corroborate this finding. In parallel, MMP-1-to-TIMP-4, MMP-2-to-TIMP-1, and MMP-2-to-TIMP-3 ratios were significantly elevated, indicating a proteolytic milieu in the myocardium, possibly causing collagen degradation. By 20 wk, many of the initial differences seen in the proteolytic ratios were not observed, with an increase in collagen compared with the 2-wk time point. Altogether, this data indicates that an imbalance in the MMP-to-TIMP ratio may occur early and potentially contribute to the early dilatation and compliance observed structurally. NEW & NOTEWORTHY In this rodent model of severe mitral regurgitation that mimics the human condition, eccentric left ventricular dilatation occurred rapidly and persisted over the 20-wk period with parallel changes in myocardial collagen and matrix metalloproteinases that may drive the extracellular matrix breakdown.

Intranasal delivery of a formulation containing stage-specific recombinant proteins of Fasciola hepatica cathepsin L5 and cathepsin B2 triggers an anti-fecundity effect and an adjuvant-mediated reduction in fluke burden in sheep.

Fasciola hepatica infection continues to be a major problem in the agriculture sector, particularly in sheep and cattle. Cathepsin L and B proteases are major components of the excretory/secretory material of the parasite, and their roles in several important aspects of parasite invasion and survival has led to their use as targets in rational vaccine design. Previous studies in rats demonstrated that the use of stage-specific antigens, cathepsin B2 and cathepsin L5, as part of a multivalent vaccine, was able to confer significant protection against challenge. In the present study, recombinant versions of cathepsin L5 and cathepsin B2 produced in yeast were used in combination to vaccinate sheep. Intramuscular and intranasal forms of administration were applied, and sheep were subsequently challenged with 150 F. hepatica metacercariae. Intramuscular vaccination was able to induce a strong systemic antibody response against both antigens, but failed to confer significant protection. Conversely, no elevated antibody response was detected against the vaccine antigens following nasal vaccination; however, a reduction in parasite egg viability (>92%) and a statistically significant (p = 0.006), predominantly adjuvant-mediated reduction in worm burdens was observed.

Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination.

No licensed vaccine is currently available for prevention of Fasciola hepatica infections. However, considering the alarming increase in drug resistance, there is an urgent need for a safe and fully effective vaccine against fasciolosis. Here, we tested if cathepsins L (FhCL3-1, FhCL3-2) and B (FhCB3) secreted by juvenile liver flukes are viable vaccine targets when delivered alone or in combination in a rat model. Since control over the early immune response is crucial for parasite's establishment in its host, it was hypothesised that targeting fluke juvenile stages may prove beneficial. Moreover, it was assumed that selected antigens will act in a cumulative manner to interfere with liver fluke migration and thereby will reduce F. hepatica infection. Recombinant FhCL3-1 and FhCL3-2 delivered alone reduced liver fluke burdens by 47 % and 63 %, respectively. A trivalent vaccine containing rFhCL3-1/CL3-2/CB3 did not increase the protective vaccine efficacy compared to the rFhCL3-2 vaccinated group (53 %), although, reductions in liver fluke wet weight (statistically significant) and liver damage score were most pronounced. Further, the highest IgG1 and IgG2a levels were seen in rFhCL3-2 vaccinated rats, the group for which the highest reduction in worm burden was demonstrated. Moreover, IgG1 and IgG2a levels in vaccinated rats were significantly elevated compared to those reported for control groups up to 4 week post-infection. While the mechanism of protection remains unknown, it appears that it depends on vaccine-induced antibodies directed against cathepsins. The obtained results imply that F. hepatica juvenile-specific cathepsins are promising vaccine candidates that induce responses that successfully target early migratory liver fluke stages. Now, the challenge is to evaluate these juvenile-specific cathepsins for use in livestock.

Sex and vaccination: Insights from female rats vaccinated with juvenile-specific proteases from Fasciola hepatica.

Most animal research is less evidence-based for females, with the majority of studies conducted on males. Since immune responses vary between males and females, sexual dimorphism in immunity contributes, among other things, to sex-based differences post-vaccination. However, the issue of sex effects in animal vaccine research is rarely considered in vaccine study design. Previously, we have evaluated the efficacy of cathepsin L3 (FhCL3-1 and FhCL3-2) and B3 proteases (FhCB3) from juvenile Fasciola hepatica as vaccines against fasciolosis in male rats. Their administration resulted in reductions in liver fluke recovery in the range of 47-63% when compared with an infection control group. Here, we investigated if the protective effect of vaccination with these proteins can also be observed for female rats. The data indicates females were not protected from F. hepatica infection when vaccinated with juvenile cathepsins. Only in the FhCL3-2 vaccinated group was a low, non-significant, reduction in worm burden observed (21%). Although liver fluke mean body lengths and wet weights were reduced in vaccinated animals when compared with the infection controls, these effects were adjuvant- not vaccine-induced, while for males changes in these parameters were related primarily to vaccination. Specific humoral responses throughout the study were evident; however, trends in antibody responses in females replicated trends observed previously for male humoral responses. Formerly, elevated levels of FhCL3-1 and FhCL3-2 specific IgG1 and IgG2a were suggested to be correlated with protection. Here, despite increased and clear responses of these antibodies, protection was not observed. Hence, in the present study the roles of IgG1 and IgG2 in liver fluke reduction are questionable. Results demonstrated in our study show that observations obtained in one sex are not always applicable to the other sex. Hopefully, the findings of the study will stimulate discussion of the issue of sex impacts on post-vaccination outcomes and will encourage researchers to consider sex in their future vaccine studies.

Pathological Remodeling of Mitral Valve Leaflets from Unphysiologic Leaflet Mechanics after Undersized Mitral Annuloplasty to Repair Ischemic Mitral Regurgitation.

Background Undersized ring annuloplasty is a commonly used surgical repair for ischemic mitral regurgitation, in which annular downsizing corrects regurgitation, but alters valve geometry and elevates tissue stresses. In this study, we investigated if unphysiological leaflet kinematics after annuloplasty might cause pathogenic biological remodeling of the mitral valve leaflets, and if preserving physiologic leaflet kinematics with a better technique can moderate such adverse remodeling. Methods and Results Twenty-nine swine were induced with ischemic mitral regurgitation, and survivors were assigned to groups: 7 underwent annuloplasty, 12 underwent annuloplasty with papillary-muscle approximation, 6 underwent papillary-muscle approximation, and 3 were sham controls. Pre-and post-surgery leaflet kinematics were measured, and valve tissue was explanted after 3 months to assess biological changes. Anterior leaflet excursion was unchanged across groups, but persistent tethering was observed with annuloplasty. Posterior leaflet was vertically immobile after annuloplasty, better mobile with the combined approach, and substantially ( P=0.0028) mobile after papillary-muscle approximation. Procollagen-1 was higher in leaflets from annuloplasty compared with the other groups. Heat shock protein-47 and lysyl oxidase were higher in groups receiving annuloplasty compared with sham. α- SMA was elevated in leaflets from animals receiving an annuloplasty, indicating activation of quiescent valve interstitial cells, paralleled by elevated transforming growth factor-ß expression. Conclusions This is the first study to demonstrate that surgical valve repairs that impose unphysiological leaflet mechanics have a deleterious, pathological impact on valve biology. Surgeons may need to consider restoring physiologic leaflet stresses as well as valve competence, while also exploring pharmacological methods to inhibit the abnormal signaling cascades.

CD39 and CD73 in the aortic valve-biochemical and immunohistochemical analysis in valve cell populations and its changes in valve mineralization.

BACKGROUND: The calcific aortic valve disease (CAVD) is a common heart pathology that involves inflammation, fibrosis, and calcification of aortic valve leaflets. All these processes could be affected by changes in the extracellular purinergic signaling that depend on the activity of ectonucleotidases, mainly ectonucleoside triphosphate diphosphohydrolase 1 (CD39, eNTPD1) and ecto-5'nucleotidase (CD73, e5NT). OBJECTIVE AND METHODS: We investigated the localization of CD39 and CD73 proteins in human noncalcified and calcified aortic valves using immunohistochemistry together with analysis of NTPDases and e5NT activities in aortic valve homogenates by analysis of substrate into product conversion by high-performance liquid chromatography. We also measured the rates of extracellular nucleotide catabolism on the surface of isolated cultured aortic valve endothelial (hAVECs) and interstitial cells (hAVICs) as well as characterized cellular CD39 and CD73 distribution. RESULTS: In noncalcified valves, CD39 and CD73 were expressed in both endothelial and interstitial cells, while in calcified valves, the expressions of CD39 and CD73 were significantly down-regulated with the exception of calcified regions where the expression of CD73 was maintained. This correlated with activities in valve homogenates. NTPDase was reduced by 35% and e5NT activity by 50% in calcified vs. noncalcified valve. CD39 and CD73 were present mainly in the cell membrane of hAVECs, but in hAVICs, these proteins were also present intracellularly. The rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis in isolated hAVECs and hAVICs were comparable. CONCLUSION: The presence of ectonucleotidases in valves and especially in aortic valve interstitial cells highlights important local role of purinergic signaling and metabolism. Changes in the local expression and hence the activity of CD39 and CD73 in calcified valves suggest their potential role in CAVD.

Metabolic pathway of 4-pyridone-3-carboxamide-1ß-d-ribonucleoside and its effects on cellular energetics.

4-pirydone-3-carboxamide-1ß-d-ribonucleoside (4PYR) is an endogenous nucleoside that could be converted to triphosphates, diphosphates, monophosphates and an analogue of NAD - 4PYRAD. Elevated level of these compounds have been reported in chronic renal failure, cancer and active HIV infection. However, little is known about the effect on cell functionality and the metabolic pathways. This study tested effects of 4PYR in different cell types on nucleotide, energy metabolism and clarified enzymes that are involved in conversions of 4PYR. We have found that human neuroblastoma cells, human malignant melanoma cells, human adipose-derived stem cells, human bone marrow-derived stem cells, human dermal microvascular endothelial cells and human embryonic kidney cells, were capable to convert 4PYR into its derivatives. This was associated with deterioration of cellular energetics. Incubation with 4PYR did not affect mitochondrial function, but decreased glycolytic rate (as measured by extracellular acidification) in endothelial cells. Silencing of adenosine kinase, cytosolic 5'-nucleotidase II and nicotinamide nucleotide adenylyltransferase 3, blocked metabolism of 4PYR. Incubation of endothelial cells with 4PYR decreased AMP deaminase activity by 40%. The main finding of this paper is that human cells (including cancer type) are capable of metabolizing 4PYR that lead to deterioration of energy metabolism, possibly as the consequence of inhibition of glycolysis. This study, it was also found that several enzymes of nucleotide metabolism could also contribute to the 4PYRconversions.

Endothelial toxicity of unusual nucleotide metabolites.

Endothelium plays a pivotal role in the vascular tone regulation, platelet aggregation, regulation of immune response, inflammation and angiogenesis and its dysfunction is an earliest event in the development of cardiovascular disease. All these processes are affected by endothelial dysfunction. Endothelial toxicity induced by metabolites present in blood is a common scenario in pathology. This involves physiological metabolites such as asymmetric dimethylarginine or homocysteine that are normally excreted by kidneys, but accumulate in pathological conditions, adversely affecting function of endothelium. Our group identified new molecule with potential endothelial toxicity: 4-pirydone-3-carboxamide-1-ß-d-ribonucleoside (4PYR). This nucleoside is most likely produced by oxidation of nicotinamide containing precursor by aldehyde oxidase. 4PYR easy crosses cell membrane and become phosphorylated inside the cell giving rise to mono-, di- and triphospates (4PYMP, 4PYDP and 4PYTP). There is considerable evidence that 4PYR is toxic in endothelium and other cell types by disrupting cell energetics evident as ATP depletion. Endothelial dysfunction in the in vitro and in vivo experiments is, however, evident only after prolonged exposure to 4PYR while acute cardiovascular effects are minor. 4PYR endothelial toxicity could be particularly important in patients with chronic renal disease where accumulation of 4PYR and its metabolites is particularly prominent. 4PYR metabolism and toxicity could be blocked by application of nucleoside transport inhibitors and we have proven efficiency of such intervention. We believe that blocking metabolism of endothelial nucleoside toxins such as 4PYR could become important strategy for endothelial targeted therapy.

4-Pyridone-3-carboxamide-1ß-D-ribonucleoside metabolism in endothelial cells and its impact on cellular energetic balance.

4-Pyridone-3-carboxamide-1ß-D-ribonucleoside (4PYR) is a naturally occurring compound related to nicotinamide that could be metabolized to mono-, di-, and triphosphates of 4PYR (4PYMP, 4PYDP, 4PYTP) and nicotinamide adenine dinucleotide (NAD) analogue (4PYRAD) in all types of cells. Previous studies demonstrated that formation of 4PYMP and 4PYTP was dependent on adenosine kinase activity. Pathway of 4PYRAD production is not yet identified, but most likely this process involves production of 4PYMP. This study aimed to evaluate influence of 4PYR on metabolism of endothelial cells and to test effect of nucleoside transport inhibitors. 4PYR was obtained by chemical synthesis. Endothelial cell line (HMEC-1) was incubated for 24 or 48 hours with 100 µM 4PYR. After incubation, cells were separated from medium and analyzed for concentrations of ATP, NAD, and 4PYR metabolites by using reversed-phase high performance liquid chromatography. We demonstrated progressive accumulation of 4PYR metabolites in endothelium that reached 33.2±0.8 nmol/mg protein for 4PYMP and 5.25±0.17 nmol/mg protein for 4PYRAD after 48-hour incubation with 4PYR. Dipyridamole protected from accumulation of 4PYR metabolites in endothelial cells. We conclude that endothelium is capable to convert 4PYR into intracellular metabolites and this causes disruption of cell energy balance. Nucleoside transport inhibition with dipyridamole could protect endothelium from this effect. This finding could be of clinical relevance in conditions associated with accumulation of 4PYR such as chronic renal disease.

Immunohistochemical and functional analysis of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73) in pig aortic valves.

Extracellular nucleotides control mechanisms such as thrombosis or inflammation that are important in several pathologies, including heart valve disease and calcification. Ectonucleoside triphosphate diphosphohydrolase 1 (eNTPD1, CD39) and ecto-5'-nucleotidase (e5NT, CD73) are ectoenzymes that convert adenosine triphosphate to adenosine diphosphate, adenosine monophosphate and finally to adenosine. Changes in activities of these enzymes influence extracellular nucleotide concentrations and therefore could be involved in valve pathology. This study aimed to analyze type of cells, specific area, level of expression and biochemical function of CD39 and CD73 in pig aortic valves. Samples were collected from aortic valves of domestic pigs. Histological sections were cut from paraffin embedded tissue blocks. Following incubation with primary antibody against CD39 or CD73, washing and secondary goat anti-rabbit secondary antibodies, slides were viewed with NanoZoomer scanner. Substantial expression CD39 and CD73 was observed in two main types of valve cells: endothelial and valve interstitial cells. Subsequently, biochemical function of CD39 and CD73 was evaluated in cells cultured from pig aortic valve. Breakdown of extracellular nucleotides added to cell medium was analyzed with high performance liquid chromatography. In the interstitial cells, the CD73 products formation was much faster than in endothelium, while for the CD39 activity this relation was opposite. Expression and high concentration of CD39 and CD73 products in endothelium are expected, but presence of CD73 in valve interstitial cells is a surprise. We conclude that CD39 and CD73 and their enzymatic activities that convert extracellular nucleotides are highly expressed and could have special function in the valve.

Geldanamycin-induced osteosarcoma cell death is associated with hyperacetylation and loss of mitochondrial pool of heat shock protein 60 (hsp60).

Osteosarcoma is one of the most malignant tumors of childhood and adolescence that is often resistant to standard chemo- and radio-therapy. Geldanamycin and geldanamycin analogs have been recently studied as potential anticancer agents for osteosarcoma treatment. Here, for the first time, we have presented novel anticancer mechanisms of geldanamycin biological activity. Moreover, we demonstrated an association between the effects of geldanamycin on the major heat shock proteins (HSPs) and the overall survival of highly metastatic human osteosarcoma 143B cells. We demonstrated that the treatment of 143B cells with geldanamycin caused a subsequent upregulation of cytoplasmic Hsp90 and Hsp70 whose activity is at least partly responsible for cancer development and drug resistance. On the other hand, geldanamycin induced upregulation of Hsp60 gene expression, and a simultaneous loss of hyperacetylated Hsp60 mitochondrial protein pool resulting in decreased viability and augmented cancer cell death. Hyperacetylation of Hsp60 seems to be associated with anticancer activity of geldanamycin. In light of the fact that mitochondrial dysfunction plays a critical role in the apoptotic signaling pathway, the presented data may support a hypothesis that Hsp60 can be another functional part of mitochondria-related acetylome being a potential target for developing novel anticancer strategies.