Positive Effects of Heme Oxygenase Upregulation on Adiposity and Vascular Dysfunction: Gene Targeting vs. Pharmacologic Therapy

Abstract

Objective: Heme oxygenase (HO-1) plays a critical role in adipogenesis and it is important to understand its function in obesity. Many studies have shown that upregulation of HO-1 can affect the biologic parameters in obesity-mediated diabetes, hypertension and vascular endothelial cell function. Thus, we aimed to explore the hypothesis that upregulation of HO-1, using a pharmacologic approach as well as gene targeting, would improve both adiposity and endothelial cell dysfunction by direct targeting of endothelial cells. Our second aim was to compare the short-term effect of a HO-1 inducer, cobalt-protoporphrin IX (CoPP), with the long-term effects of gene targeted therapy on vascular and adipocyte stem cells in obese mice. Method: We examined the effect of CoPP on fat pre-adipocytes and mesenchymal stem cells (MSC) in mice fed a high-fat diet (HFD). We also used a lentiviral construct that expressed heme oxygenase (HO-1) that was under the control of an endothelium specific promoter, vascular endothelium cadherin (VECAD) heme oxygenase (VECAD-HO-1). We targeted endothelial cells using vascular endothelium cadherin/green fluorescent protein fusion construct (VECAD-GFP) as the control. Conditioned media (CM) from endothelial cells (EC) was added to fat derived adipocytes. Additionally, we treated renal interlobar arteries with phenylephrine and dosed cumulative increments of acetylcholine both with and without exposure to CoPP. We did the same vascular reactivity experiments with VECAD-HO-1 lentiviral construct compared to the control. Results: CoPP improved vascular reactivity and decreased adipogenesis compared to the control. MSCs exposed to CM from EC transfected with VECAD-HO-1 showed decreased adipogenesis, smaller lipid droplet size and decreased PPAR-γ, C/EBP and increased Wnt 10b compared to the control. HO-1 upregulation had a direct effect on reducing adipogenesis. This effect was blocked by tin mesoporphrin (SnMP). EC treated with VECAD-HO-1 expressed lower levels of ICAM and VCAM compared to the control, suggesting improved EC function. This also improved ACH induced vascular reactivity. These effects were also reversed by SnMP. The effect of viral transfection was much more specific and sustained than the effects of pharmacologic therapy, CoPP. Conclusion: This study demonstrates that a pharmacological inducer of HO-1 such as CoPP improves endothelial cell function while dampening adipogenesis, but long-term HO-1 expression by direct targeting of endothelial cells by gene transfer therapy may offer a more specific and ideal solution. This was evidenced by smaller healthier adipocytes that had improved insulin sensitivity, suggesting increased adiponectin levels. HO-1 upregulation reestablished the “crosstalk” between perivascular adipose tissue and the vascular system that was lost in the chronic inflammatory state of obesity. This study demonstrates that gene targeting of EC may well be the future direction in treating obesity induced EC dysfunction, with the finding that targeting the vasculature had a direct and sustained effect on adipogenesis.

Adipocyte Specific HO-1 Gene Therapy Is Effective in Antioxidant Treatment of Insulin Resistance and Vascular Function in an Obese Mice Model

“…In this study, we demonstrate that adipocyte-specific HO-1 expression improves adipocyte hypertrophy by decreasing cell size (Figure 1); the progression of obesity is tightly associated with increased adipocyte hypertrophy [24,25,40]. An increase in adipocyte cell size is negatively correlated with adiponectin levels [22].…”

“…ROS plays a major role in reprogramming a normal adipocyte phenotype to inflamed adipocytes, resulting in adiposopathy [22][23][24]. Affected adipocytes proceed to terminal differentiation and inflammation, resulting in adipocyte dysfunction [23,25]. Obese animal models have low levels of HO-1 and are therefore more susceptible to oxidative stress and cytotoxicity caused by a build-up of heme, a pro-oxidant, and ROS [1,24,26].…”

Heme Oxygenase Dependent Bilirubin Generation in Vascular Cells: A Role in Preventing Endothelial Dysfunction in Local Tissue Microenvironment?

“…The same group, more recently focused on long-term endothelial HMOX1 activation in mice fed a HFD demonstrating the gene therapy to increase expression of HMOX1, reduced ICAM and VCAM expression, decreased serum markers of inflammation, such as IL-1 and TNFα, reduced the size of adipocytes and down-regulated PPARγ. Furthermore, these effects were prevented by using a specific inhibitor of HMOX1 activity, indirectly suggesting that the ability to restore a proper crosstalk between the vasculature and adipocytes depends on the metabolic products of endothelial HMOX1 activity (Peterson et al, 2019). Together these findings demonstrate that endothelial activation of HMOX1 could play an important role in the treatment/prevention of obesity, and further highlight the importance of AMPK-dependent signaling in mediating the anti-obesogenic effects of bilirubin.…”

“…AngII, angiotensin II; BRT, bilirubin ditaurate; BLVRA, biliverdin reductase; CAD, coronary artery disease; CO, carbon monoxide; CoPPIX, cobalt protoporphyrin IX; CRC, colorectal cancer patients; db/db, diabetic mice; DIO, diet-induced obese; DM, diabetes mellitus; DSS, dextran sodium sulfate; EAE, experimental autoimmune encephalomyelitis; FMD, fasting-mimicking diet; FVB, friend leukemia virus B; GI, gastro-intestinal; GS, Gilbert Syndrome; HFD, high fat diet; IRI, ischemia/reperfusion injury; Ldlr −/− , low-density lipoprotein receptor-deficient mice; MCAO, middle cerebral artery occlusion; MS, multiple sclerosis; OVA, ovalbumin; PMBCs, peripheral blood mononuclear cells; RA, rheumatoid arthritis; SHR, spontaneously hypertensive rat; SJL, Swiss Jim Lambert mice; SLE, systemic lupus erythematosus; SnPP, tin protoporphyrin IX; STS, short-term starvation; STZ, streptozocin; UCB, unconjugated bilirubin; UTG1A1, uridine diphpspho-glucuronosyl transferase 1A1; ZnPP, zinc protoporphyrin IX; WKI, Wistar Kyoto rat. cEPCs 10-20 mg/dl Progenitor endothelial cells induced to proliferate when exposed to 5 mg/dl bilirubin; higher concentrations (up to 20 mg/dl) induce cell death Jabarpour et al, 2018 EA.hy926 0.5-100 µM Exogenous bilirubin increases endothelial antioxidant activity as well as HMOX1-dependent bilirubin generation Ziberna et al, 2016 Commercially EC Not evaluated EC-transfected with HMOX1 release substances that increase healthy adipocytes Peterson et al, 2019 Human and non-human EC HUVECs and H5V cells 0.015 µM < Bf < 0.030 µM UCB, at clinically relevant concentrations, limits over-expression of adhesion molecules and inhibits PMN-endothelial adhesion induced by pro-inflammatory cytokine TNFα, even though UCB itself does not alter expression of these adhesion molecules. Inhibition NF-kappaB transduction pathway Mazzone et al, 2009a,b HAECs and mAECs Not evaluated In HAECs and in primary mAECs from HMOX1 +/+ and HMOX1 −/− , SDF-1 (100-200 ng/ml) favors angiogenesis through the induction of HMOX1 Deshane et al, 2007 (Continued) ( Deshane et al, 2007).…”

Involvement of HO-1 and Autophagy in the Protective Effect of Magnolol in Hepatic Steatosis-Induced NLRP3 Inflammasome Activation In Vivo and In Vitro

“…We showed that MG administration increased the expression of HO-1 and SOD2 by promoting the translocation of Nrf2 into the nucleus. The induction of HO-1 and SOD2 led to the alleviation of oxidative stress and conferred resistance to inflammatory insults [ 6 , 43 ]. HO-1 is the rate-limiting enzyme of heme catabolism, which catalyzes the cellular heme to carbon monoxide (CO), biliverdin, and free iron and exerts significant anti-inflammatory effects [ 44 ].…”