Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models have contributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of these pathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiac diseases, and the same pathology have been successfully recreated in different species, including small and big animal models of disease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficult to match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the human complication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specialized personnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selected that best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expanding and occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible to devise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases.

Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.

The mechanisms that permit adult tissues to regenerate when injured are not well understood. Initiation of liver regeneration requires the injury-related cytokines, tumor necrosis factor (TNF) α and interleukin (IL) 6, and involves the activation of cytokine-regulated transcription factors such as NF-κβ and STAT3. During regeneration, TNFα and IL-6 promote hepatocyte viability, as well as proliferation, because interventions that inhibit either cytokine not only block hepatocyte DNA synthesis, but also increase liver cell death. These observations suggest that the cytokines induce hepatoprotective factors in the regenerating liver. Given evidence that nitric oxide can prevent TNF-mediated activation of the pro-apoptotic protease caspase 3 and protect hepatocytes from cytokine-mediated death, cytokine-inducible nitric oxide synthase (iNOS) may be an important hepatoprotective factor in the regenerating liver. In support of this hypothesis we report that the hepatocyte proliferative response to partial liver resection is severely inhibited in transgenic mice with targeted disruption of the iNOS gene. Instead, partial hepatectomy is followed by increased caspase 3 activity, hepatocyte death, and liver failure, despite preserved induction of TNFα, IL-6, NF-κβ, and STAT3. These results suggest that during successful tissue regeneration, injury-related cytokines induce factors, such as iNOS and its product, NO, that protect surviving cells from cytokine-mediated death.

We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-alpha and IFN-gamma. Since TNF-alpha and IFN-gamma stimulate cells in part by activating NF-kappaB and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-kappaB co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that IRF-1 and NF-kappaB interact in stimulated but not resting cells. Super-shift experiments show that IRF-1 and NF-kappaB interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-kappaB proteins in vivo. We next suggested that this interaction between IRF-1 and NF-kappaB bends the DNA of the iNOS promoter region. Using a cyclization assay, we demonstrate that nuclear extracts from stimulated cells accelerate the rate of conversion of a linear to circular DNA, compared to extracts from resting cells. However, stimulated nuclear extracts cannot affect the rate of cyclization of a promoter with a mutant IRE or kappaB site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-kappaB cannot induce cyclization. We conclude that IRF-1 and NF-kappaB interact in vivo, and that this interaction physically bends the indicible nitric oxide synthase promoter DNA. This interaction may explain the mechanism by which IFN-gamma synergistically augments inducible nitric oxide synthase transcription.

Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-β ₁ expression and endothelial dysfunction. Smad proteins are a key step in TGF-β–initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-β–dependent gene expression. We show that NO inhibits TGF-β/Smad–regulated gene transactivation in a cGMP-dependent manner. NO effects were mimicked by a soluble analogue of cGMP. Inhibition of cGMP-dependent protein kinase 1 (PKG-1) or overexpression of dominant-negative PKG-1α suppressed NO/cGMP inhibition of TGF-β–induced gene expression. Inversely, overexpression of PKG-1α catalytic subunit blocked TGF-β–induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas N ^G -nitro- l -arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-β. Aortas from endothelial NO synthase–deficient mice showed enhanced basal TGF-β ₁ and collagen type I expression; endothelial cells from these animals showed increased Smad phosphorylation and transcriptional activity. Proteasome inhibitors prevented the inhibitory effect of NO on TGF-β signaling. NO reduced the metabolic life of ectopically expressed Smad2 and enhanced its ubiquitination. Taken together, these results suggest that the endothelial NO/cGMP/PKG pathway interferes with TGF-β/Smad2 signaling by directing the proteasomal degradation of activated Smad.

The host response to Coxsackievirus infection is complex, including T lymphocytes, B lymphocytes, natural killer cells, and macrophages. Although Coxsackievirus infection induces expression of inducible nitric oxide synthase (NOS2; EC1.14.13.39) in macrophages, the precise role of NOS2 in the host response to Coxsackievirus myocarditis has been unclear. We show, by using mice homozygous for a disrupted NOS2 allele, that Coxsackievirus replicates to higher titers in NOS2^−/−mice, that the host lacking NOS2 clears virus more slowly than the wild-type host, and that myocarditis is much more severe in infected NOS2^−/−mice. These data show that NOS2 is crucial for the host response to Coxsackievirus in the mouse.