Anupam Agarwal

Heme Oxygenase-1 Modulates Early Inflammatory Responses: Evidence from the Heme Oxygenase-1-Deficient Mouse

Induction of heme oxygenase-1 (HO-1) is protective in tissue injury in models of allograft rejection and vascular inflammation through either prevention of oxidative damage or via immunomodulatory effects. To examine the specific role of HO-1 in modulating the immune response, we examined the differences in immune phenotype between HO-1 knockout (HO-1(-/-)) and wild-type (HO-1(+/+)) mice. Consistent with previous findings, marked splenomegaly and fibrosis were observed in HO-1(-/-) mice. The lymph nodes of HO-1-deficient mice demonstrated a relative paucity of CD3- and B220-positive cells, but no such abnormalities were observed in the thymus. Flow cytometric analysis of isolated splenocytes demonstrated no differences in the proportions of T lymphocytes, B lymphocytes or monocytes/macrophages between the HO-1(-/-) and HO-1(+/+) mice. Significantly higher baseline serum IgM levels were observed in HO-1(-/-) versus HO-1(+/+) mice. Under mitogen stimulation with either lipopolysaccharide or anti-CD3/anti-CD28, HO-1(-/-) splenocytes secreted disproportionately higher levels of pro-inflammatory Th1 cytokines as compared to those from HO-1(+/+) mice. These findings demonstrate significant differences in the immune phenotype between the HO-1(-/-) and the HO-1(+/+) mice. The absence of HO-1 correlates with a Th1-weighted shift in cytokine responses suggesting a general pro-inflammatory tendency associated with HO-1 deficiency.

Nitrated Fatty Acids: Endogenous Anti-inflammatory Signaling Mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-γ activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-κB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-κB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-α-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.

The Synthetic Triterpenoids, CDDO and CDDO-Imidazolide, Are Potent Inducers of Heme Oxygenase-1 and Nrf2/ARE Signaling

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its derivative 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are multifunctional molecules with potent antiproliferative, differentiating, and anti-inflammatory activities. At nanomolar concentrations, these agents rapidly increase the expression of the cytoprotective heme oxygenase-1 (HO-1) enzyme in vitro and in vivo. Transfection studies using a series of reporter constructs show that activation of the human HO-1 promoter by the triterpenoids requires an antioxidant response element (ARE), a cyclic AMP response element, and an E Box sequence. Inactivation of one of these response elements alone partially reduces HO-1 induction, but mutations in all three sequences entirely eliminate promoter activity in response to the triterpenoids. Treatment with CDDO-Im also elevates protein levels of Nrf2, a transcription factor previously shown to bind ARE sequences, and increases expression of a number of antioxidant and detoxification genes regulated by Nrf2. The triterpenoids also reduce the formation of reactive oxygen species in cells challenged with tert-butyl hydroperoxide, but this cytoprotective activity is absent in Nrf2 deficient cells. These studies are the first to investigate the induction of the HO-1 and Nrf2/ARE pathways by CDDO and CDDO-Im, and our results suggest that further in vivo studies are needed to explore the chemopreventive and chemotherapeutic potential of the triterpenoids.

Sepsis and Acute Kidney Injury

Sepsis is a severe and dysregulated inflammatory response to infection characterized by end-organ dysfunction distant from the primary site of infection. Development of acute kidney injury (AKI) during sepsis increases patient morbidity, predicts higher mortality, has a significant effect on multiple organ functions, is associated with an increased length of stay in the intensive care unit, and hence consumes considerable healthcare resources. When compared with AKI of nonseptic origin, septic AKI is characterized by a distinct pathophysiology and therefore requires a different approach. Despite impressive advances in several fields of medicine, the pathophysiology, diagnostic procedures, and appropriate therapeutic interventions in sepsis are still highly debatable. Numerous immunomodulatory agents showing promise in preclinical studies fail to reduce the overwhelmingly high mortality rate of sepsis and provoke AKI when compared with other critically ill patients. Major impediments to progress in understanding, early diagnosis, and application of appropriate therapeutic modalities in sepsis-induced AKI include limited histopathologic information, few animal models that closely mimic human sepsis, and a relative shortage of specific diagnostic tools. Here we discuss the most recent advances in understanding the fundamental mechanisms of sepsis-induced AKI, characteristics of relevant animal models available, and potential therapies.

Mechanism of Action of Sulforaphane: Inhibition of p38 Mitogen-Activated Protein Kinase Isoforms Contributing to the Induction of Antioxidant Response Element–Mediated Heme Oxygenase-1 in Human Hepatoma HepG2 Cells

Exposure of sulforaphane to HepG2 cells increased heme oxygenase-1 (HO-1) expression by activating antioxidant response element (ARE) through induction of Nrf2 and suppression of Kelch-like ECH-associated protein 1 (Keap1). Using human HO-1 promoter reporter plasmids and ChIP assay, we have identified that sulforaphane transcriptionally activated the upstream ARE-rich enhancer region, located at -9.0 kb upstream human HO-1 promoter. Induction of HO-1 by sulforaphane was attenuated by overexpression of mutant Nrf2 plasmid in HepG2 cells and totally abolished in Nrf2 knockout mouse embryonic keratinocytes and fibroblasts. Overexpression of individual p38 mitogen-activated protein (MAP) kinase (MAPK) isoforms also suppressed constitutive as well as sulforaphane- or Nrf2-induced ARE-dependent gene expression. Among the upstream kinases, although MKK3 was not involved in suppression of ARE by any of p38 MAPK isoforms, MKK6 selectively suppressed ARE by p38 gamma or p38 delta, but not by p38 alpha or p38 beta. Importantly, sulforaphane not only activated MAP/extracellular signal-regulated kinase (ERK) kinases 1/2 and ERK1/2, but also strongly suppressed anisomycin-induced activation of p38 MAPK isoforms by blocking phosphorylation of upstream kinases, MKK3/6. Finally, we found that stimulation of p38 MAPK isoforms phosphorylated purified Nrf2 protein and caused an increase in the interaction between Nrf2 and Keap1 in vitro and the suppression of Nrf2 translocation into the nucleus. Collectively, our results indicate that transcriptional activation of Nrf2/ARE is critical in sulforaphane-mediated induction of HO-1, which can be modulated in part by the blockade of p38 MAPK signaling pathway. In addition, our study shows that p38 MAPK can phosphorylate Nrf2 and promotes the association between Nrf2 and Keap1 proteins, thereby potentially inhibiting nuclear translocation of Nrf2.

Heme Oxygenase-1 and Carbon Monoxide in Vascular Pathobiology Focus on Angiogenesis

Angiogenesis involves the formation of new blood vessels and is critical for fundamental events such as development and repair after injury. Perturbances in angiogenesis contribute to the pathogenesis of diverse clinical conditions including cancer, complications of diabetes mellitus, ischemia/reperfusion injury of the heart and other organs, and preeclampsia, as well as a number of inflammatory disorders. Recent work has identified heme oxygenase-1 and its gaseous product, carbon monoxide, to possess potent proangiogenic properties in addition to well-recognized antiinflammatory, antioxidant, and antiapoptotic effects. Angiogenic factors, such as vascular endothelial growth factor and stromal cell-derived factor-1, mediate their proangiogenic effects through induction of heme oxygenase-1, making it an attractive target for therapeutic intervention. This review will provide an overview of the role of heme oxygenase-1 and carbon monoxide in angiogenesis.

Stromal cell–derived factor 1 promotes angiogenesis via a heme oxygenase 1–dependent mechanism

Stromal cell–derived factor 1 (SDF-1) plays a major role in the migration, recruitment, and retention of endothelial progenitor cells to sites of ischemic injury and contributes to neovascularization. We provide direct evidence demonstrating an important role for heme oxygenase 1 (HO-1) in mediating the proangiogenic effects of SDF-1. Nanomolar concentrations of SDF-1 induced HO-1 in endothelial cells through a protein kinase C ζ–dependent and vascular endothelial growth factor–independent mechanism. SDF-1–induced endothelial tube formation and migration was impaired in HO-1–deficient cells. Aortic rings from HO-1−/− mice were unable to form capillary sprouts in response to SDF-1, a defect reversed by CO, a byproduct of the HO-1 reaction. Phosphorylation of vasodilator-stimulated phosphoprotein was impaired in HO-1−/− cells, an event that was restored by CO. The functional significance of HO-1 in the proangiogenic effects of SDF-1 was confirmed in Matrigel plug, wound healing, and retinal ischemia models in vivo. The absence of HO-1 was associated with impaired wound healing. Intravitreal adoptive transfer of HO-1–deficient endothelial precursors showed defective homing and reendothelialization of the retinal vasculature compared with HO-1 wild-type cells following ischemia. These findings demonstrate a mechanistic role for HO-1 in SDF-1–mediated angiogenesis and provide new avenues for therapeutic approaches in vascular repair.

Heme Oxygenase-1 and the Vascular Bed: From Molecular Mechanisms to Therapeutic Opportunities

Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.

Identification of a microRNA signature in renal fibrosis: role of miR-21

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-β1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-β1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.

Heme Oxygenase-1-derived Carbon Monoxide Induces the Mycobacterium tuberculosis Dormancy Regulon

The mechanisms that allow Mycobacterium tuberculosis (Mtb) to persist in human tissue for decades and to then abruptly cause disease are not clearly understood. Regulatory elements thought to assist Mtb to enter such a state include the heme two-component sensor kinases DosS and DosT and the cognate response regulator DosR. We have demonstrated previously that O2, nitric oxide (NO), and carbon monoxide (CO) are regulatory ligands of DosS and DosT. Here, we show that in addition to O2 and NO, CO induces the complete Mtb dormancy (Dos) regulon. Notably, we demonstrate that CO is primarily sensed through DosS to induce the Dos regulon, whereas DosT plays a less prominent role. We also show that Mtb infection of macrophage cells significantly increases the expression, protein levels, and enzymatic activity of heme oxygenase-1 (HO-1, the enzyme that produces CO), in an NO-independent manner. Furthermore, exploiting HO-1+/+ and HO-1-/- bone marrow-derived macrophages, we demonstrate that physiologically relevant levels of CO induce the Dos regulon. Finally, we demonstrate that increased HO-1 mRNA and protein levels are produced in the lungs of Mtb-infected mice. Our data suggest that during infection, O2, NO, and CO are being sensed concurrently rather than independently via DosS and DosT. We conclude that CO, a previously unrecognized host factor, is a physiologically relevant Mtb signal capable of inducing the Dos regulon, which introduces a new paradigm for understanding the molecular basis of Mtb persistence.