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1.
Am J Physiol Heart Circ Physiol ; 323(4): H670-H687, 2022 10 01.
Article in English | MEDLINE | ID: mdl-35985007

ABSTRACT

Endotoxemia elicits a multiorgan inflammatory response that results in cardiac dysfunction and often leads to death. Inflammation-induced metabolism of endogenous N-3 and N-6 polyunsaturated fatty acids generates numerous lipid mediators, such as epoxy fatty acids (EpFAs), which protect the heart. However, EpFAs are hydrolyzed by soluble epoxide hydrolase (sEH), which attenuates their cardioprotective actions. Global genetic disruption of sEH preserves EpFA levels and attenuates cardiac dysfunction in mice following acute lipopolysaccharide (LPS)-induced inflammatory injury. In leukocytes, EpFAs modulate the innate immune system through the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. However, the mechanisms by which both EpFAs and sEH inhibition exert their protective effects in the cardiomyocyte are still elusive. This study investigated whether cardiomyocyte-specific sEH disruption attenuates inflammation and cardiac dysfunction in acute LPS inflammatory injury via modulation of the NLRP3 inflammasome. We use tamoxifen-inducible CreER recombinase technology to target sEH genetic disruption to the cardiomyocyte. Primary cardiomyocyte studies provide mechanistic insight into inflammasome signaling. For the first time, we demonstrate that cardiomyocyte-specific sEH disruption preserves cardiac function and attenuates inflammatory responses by limiting local cardiac inflammation and activation of the systemic immune response. Mechanistically, inhibition of cardiomyocyte-specific sEH activity or exogenous EpFA treatment do not prevent upregulation of NLRP3 inflammasome machinery in neonatal rat cardiomyocytes. Rather, they limit downstream activation of the pathway leading to release of fewer chemoattractant factors and recruitment of immune cells to the heart. These data emphasize that cardiomyocyte sEH is vital for mediating detrimental systemic inflammation.NEW & NOTEWORTHY The cardioprotective effects of genetic disruption and pharmacological inhibition of sEH have been demonstrated in a variety of cardiac disease models, including acute LPS inflammatory injury. For the first time, it has been demonstrated that sEH genetic disruption limited to the cardiomyocyte profoundly preserves cardiac function and limits local and systemic inflammation following acute LPS exposure. Hence, cardiomyocytes serve a critical role in the innate immune response that can be modulated to protect the heart.


Subject(s)
Heart Diseases , Myocytes, Cardiac , Animals , Chemotactic Factors/therapeutic use , Epoxide Hydrolases/genetics , Fatty Acids/metabolism , Fatty Acids, Unsaturated/therapeutic use , Inflammasomes , Inflammation/drug therapy , Lipopolysaccharides/pharmacology , Mice , Mice, Knockout , Myocytes, Cardiac/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Rats , Recombinases/therapeutic use , Tamoxifen/therapeutic use
2.
PLoS One ; 7(2): e31576, 2012.
Article in English | MEDLINE | ID: mdl-22348110

ABSTRACT

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.


Subject(s)
Cell Membrane Permeability , DNA Repair , DNA, Viral/isolation & purification , HIV Infections/therapy , HIV-1/genetics , Recombinases/administration & dosage , Cloning, Molecular , DNA, Viral/metabolism , Escherichia coli/genetics , HIV Long Terminal Repeat , HeLa Cells , Humans , Recombinant Proteins/therapeutic use , Recombinases/metabolism , Recombinases/therapeutic use
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