Sodium-glucose cotransporter 2 inhibitor Dapagliflozin attenuates diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus type 2 (DM2) is a risk factor for developing heart failure but there is no specific therapy for diabetic heart disease. Sodium glucose transporter 2 inhibitors (SGLT2I) are recently developed diabetic drugs that primarily work on the kidney. Clinical data describing the cardiovascular benefits of SGLT2Is highlight the potential therapeutic benefit of these drugs in the prevention of cardiovascular events and heart failure. However, the underlying mechanism of protection remains unclear. We investigated the effect of Dapagliflozin-SGLT2I, on diabetic cardiomyopathy in a mouse model of DM2. METHODS: Cardiomyopathy was induced in diabetic mice (db/db) by subcutaneous infusion of angiotensin II (ATII) for 30 days using an osmotic pump. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water. Male homozygous, 12-14 weeks old WT or db/db mice (n = 4-8/group), were used for the experiments. Isolated cardiomyocytes were exposed to glucose (17.5-33 mM) and treated with Dapagliflozin in vitro. Intracellular calcium transients were measured using a fluorescent indicator indo-1. RESULTS: Angiotensin II infusion induced cardiomyopathy in db/db mice, manifested by cardiac hypertrophy, myocardial fibrosis and inflammation (TNFα, TLR4). Dapagliflozin decreased blood glucose (874 ± 111 to 556 ± 57 mg/dl, p < 0.05). In addition it attenuated fibrosis and inflammation and increased the left ventricular fractional shortening in ATII treated db/db mice. In isolated cardiomyocytes Dapagliflozin decreased intracellular calcium transients, inflammation and ROS production. Finally, voltage-dependent L-type calcium channel (CACNA1C), the sodium-calcium exchanger (NCX) and the sodium-hydrogen exchanger 1 (NHE) membrane transporters expression was reduced following Dapagliflozin treatment. CONCLUSION: Dapagliflozin was cardioprotective in ATII-stressed diabetic mice. It reduced oxygen radicals, as well the activity of membrane channels related to calcium transport. The cardioprotective effect manifested by decreased fibrosis, reduced inflammation and improved systolic function. The clinical implication of our results suggest a novel pharmacologic approach for the treatment of diabetic cardiomyopathy through modulation of ion homeostasis.

PPARδ binding to heme oxygenase 1 promoter prevents angiotensin II-induced adipocyte dysfunction in Goldblatt hypertensive rats.

OBJECTIVE: Renin-angiotensin system (RAS) regulates adipogenic response with adipocyte hypertrophy by increasing oxidative stress. Recent studies have shown the role of peroxisome proliferator-activated receptor-δ (PPARδ) agonist in attenuation of angiotensin II-induced oxidative stress. The aim of this study was to explore a potential mechanistic link between PPARδ and the cytoprotective enzyme heme oxygenase-1 (HO-1) and to elucidate the contribution of HO-1 to the adipocyte regulatory effects of PPARδ agonism in an animal model of enhanced RAS, the Goldblatt 2 kidney 1 clip (2K1C) model. METHOD: We first established a direct stimulatory effect of the PPARδ agonist (GW 501516) on the HO-1 gene by demonstrating increased luciferase activity in COS-7 cells transfected with a luciferase-HO-1 promoter construct. Sprague-Dawley rats were divided into four groups: sham-operated animals, 2K1C rats and 2K1C rats treated with GW 501516, in the absence or presence of the HO activity inhibitor, stannous mesoporphyrin (SnMP). RESULTS: 2K1C animals had increased visceral adiposity, adipocyte hypertrophy, increased inflammatory cytokines, increased circulatory and adipose tisssue levels of renin and Ang II along with increased adipose tissue gp91 phox expression (P<0.05) when compared with sham-operated animals. Treatment with GW 501516 increased adipose tissue HO-1 and adiponectin levels (P<0.01) along with enhancement of Wnt10b and ß-catenin expression. HO-1 induction was accompanied by the decreased expression of Wnt5b, mesoderm specific transcript (mest) and C/EBPα levels and an increased number of small adipocytes (P<0.05). These effects of GW501516 were reversed in 2K1C animals exposed to SnMP (P<0.05). CONCLUSION: Taken together, our study demonstrates, for the first time, that increased levels of Ang II contribute towards adipose tissue dysregulation, which is abated by PPARδ-mediated upregulation of the heme-HO system. These findings highlight the pivotal role and symbiotic relationship of HO-1, adiponectin and PPARδ in the regulation of metabolic homeostasis in adipose tissues.

Apolipoprotein A-I mimetic peptide L-4F prevents myocardial and coronary dysfunction in diabetic mice.

Diabetes is a major health problem associated with adverse cardiovascular outcomes. The apolipoprotein A-I mimetic peptide L-4F is a putative anti-diabetic drug, has antioxidant and anti-inflammatory proprieties and improves endothelial function. In obese mice L-4F increases adiponectin levels, improving insulin sensitivity, and reducing visceral adiposity. We hypothesized that the pleiotropic actions of L-4F can prevent heart and coronary dysfunction in a mouse model of genetically induced Type II diabetes. We treated db/db mice with either L-4F or vehicle for 8 weeks. Trans-thoracic echocardiography was performed; thereafter, isolated hearts were subjected to ischemia/reperfusion (IR). Glucose, insulin, adiponectin, and pro-inflammatory cytokines (IL-1ß, TNF-α, MCP-1) were measured in plasma and HO-1, pAMPK, peNOS, iNOS, adiponectin, and superoxide in cardiac tissue. In db/db mice L-4F decreased accumulation of subcutaneous and total fat, and increased insulin sensitivity and adiponectin levels while lowering inflammatory cytokines (P < 0.05). L-4F normalized in vivo left ventricular (LV) function of db/db mice, increasing (P < 0.05) fractional shortening and decreasing (P < 0.05) LV dimensions. In I/R experiments, L-4F prevented coronary microvascular resistance from increasing and LV function from deteriorating in the db/db mice. These changes were associated with increased cardiac expression of HO-1, pAMPK, peNOS, and adiponectin and decreased levels of superoxide and iNOS (P < 0.01). In the present study we showed that L-4F prevented myocardial and coronary functional abnormalities in db/db mice. These effects were associated with stimulation of HO-1 resulting in increased levels of anti-inflammatory, anti-oxidative, and vasodilatatory action through a mechanism involving increased levels of adiponectin, pAMPK, and peNOS.

Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats.

Cytochrome P-450-dependent metabolites of arachidonic acid (AA) increased in the kidneys of young, spontaneously hypertensive rats (SHRs) during the period of rapid elevation of blood pressure (BP) but not in adult SHRs or in Wistar Kyoto rats (WKYs) with normal BP. Treatment of SHRs and WKYs with stannous chloride (SnCl2), which selectively depletes renal cytochrome P-450, restored BP to normal, coincident with a natriuresis, in young but not in adult SHRs and did not affect either BP or sodium excretion in WKYs. Depletion of renal cytochrome P-450 was associated with decreased generation of these AA metabolites only in young SHRs. The antihypertensive effect of SnCl2 in young SHRs was greatly reduced by prevention of its cytochrome P-450-depleting action.

CYP-450 Epoxygenase Derived Epoxyeicosatrienoic Acid Contribute To Reversal of Heart Failure in Obesity-Induced Diabetic Cardiomyopathy via PGC-1 α Activation.

We have previously shown that an Epoxyeicosatrienoic Acid (EET) -agonist has pleiotropic effects and reverses cardiomyopathy by decreasing inflammatory molecules and increasing antioxidant signaling. We hypothesized that administration of an EET agonist would increase Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α), which controls mitochondrial function and induction of HO-1 and negatively regulates the expression of the proinflammatory adipokines CCN3/NOV in cardiac and pericardial tissues. This pathway would be expected to further improve left ventricular (LV) systolic function as well as increase insulin receptor phosphorylation. Measurement of the effect of an EET agonist on oxygen consumption, fractional shortening, blood glucose levels, thermogenic and mitochondrial signaling proteins was performed. Control obese mice developed signs of metabolic syndrome including insulin resistance, hypertension, inflammation, LV dysfunction, and increased NOV expression in pericardial adipose tissue. EET agonist intervention decreased pericardial adipose tissue expression of NOV, while normalized FS, increased PGC-1α, HO-1 levels, insulin receptor phosphorylation and improved mitochondrial function, theses beneficial effect were reversed by deletion of PGC-1α. These studies demonstrate that an EET agonist increases insulin receptor phosphorylation, mitochondrial and thermogenic gene expression, decreased cardiac and pericardial tissue NOV levels, and ameliorates cardiomyopathy in an obese mouse model of the metabolic syndrome.

Effect of heme arginate administration on blood pressure in spontaneously hypertensive rats.

Cytochrome P450 content and activities are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared with those of normotensive, Wistar-Kyoto (WKY), control rats during the period of rapid elevation of blood pressure. We studied the effect of heme arginate, a potent inducer of heme oxygenase (EC 1.14.99.3), on microsomal cytochrome P450 levels and activities and blood pressure in SHR at 7 wk of age. Administration of heme arginate (15 mg/kg body weight for 4 d) resulted in a marked decrease in blood pressure from 156.3 +/- 4.7 to 129.8 +/- 4.5 mm Hg (P less than 0.001), whereas blood pressure in SHR receiving the vehicle control was not affected. The blood pressure of age-matched WKY was not affected by heme arginate. Heme oxygenase activity increased in both hepatic and renal microsomes of SHR and WKY by two- to four-fold after treatment with heme arginate. Maximal increase of heme oxygenase mRNA occurred 5-7 h after the last injection of heme arginate and returned to control levels after 24 h. The increase in heme oxygenase activity was associated with a parallel decrease in cytochrome P450 content and in the activity of cytochrome P450 omega/omega-1 arachidonate hydroxylases in kidneys of SHR. It is postulated that heme arginate treatment resulted in induction of heme oxygenase which consequently led to a diminution of cytochrome P450, especially the arachidonate omega/omega-1 hydroxylases leading to a marked decrease in 19-hydroxyeicosatetraenoic acid (HETE) and 20-HETE. The effect of heme arginate on blood pressure may be mediated via these biochemical events inasmuch as both 19-HETE and 20-HETE produced by the kidney may promote hypertension by causing vasoconstriction and sodium retention.

Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors.

Rat renal interlobar arteries express heme oxygenase 2 (HO-2) and manufacture carbon monoxide (CO), which is released into the headspace gas. CO release falls to 30% and 54% of control, respectively, after inhibition of HO activity with chromium mesoporphyrin (CrMP) or of HO-2 expression with antisense oligodeoxynucleotides (HO-2 AS-ODN). Patch-clamp studies revealed that CrMP decreases the open probability of a tetraethylammonium-sensitive (TEA-sensitive) 105 pS K channel in interlobar artery smooth muscle cells, and that this effect of CrMP is reversed by CO. Assessment of phenylephrine-induced tension development revealed reduction of the EC(50) in vessels treated with HO-2 AS-ODN, CrMP, or TEA. Exogenous CO greatly minimized the sensitizing effect on agonist-induced contractions of agents that decrease vascular CO production, but not the sensitizing effect of K channel blockade with TEA. Collectively, these data suggest that vascular CO serves as an inhibitory modulator of vascular reactivity to vasoconstrictors via a mechanism that involves a TEA-sensitive K channel.

Metabolic syndrome: psychosocial, neuroendocrine, and classical risk factors in type 2 diabetes.

This article summarizes some aspects of stress in the metabolic syndrome at the psychosocial, tissue, and cellular levels. The metabolic syndrome is a valuable research concept for studying population health and social-biological translation. The cluster of cardiovascular risk factors labeled the metabolic syndrome is linked with low socioeconomic status. Systematic differences in diet and physical activity contribute to social patterning of the syndrome. In addition, psychosocial factors including chronic work stress are linked with its development. Psychosocial factors could lead to metabolic perturbations and increase cardiovascular risk via activation of neuroendocrine responses, for example, in the autonomic nervous system and in several hormonal pathways. High glucocorticoid levels will promote lipid storage in visceral rather than subcutaneous adipose tissue. Adipocytes secrete several proinflammatory cytokines, which considered major contributors to increase in oxidants and cell injury. Upregulation of heme oxygenase 1 (HO-1) and peroxidase in the early development of diabetes produces a decrease in oxidative-mediated injury. Increased HO activity is associated with a significant decrease in superoxide, endothelial cell shedding and blood pressure. Finally, it is proposed that overexpression of glutathione peroxidase in beta cells may protect beta cell deterioration from oxidative stress during development of diabetes and hyperglycemia and this may result in attenuation of beta cell failure. If this proves to be the case, then the scene will be set to develop glutathione peroxidase mimetics for use in preclinical and clinical trials.

Treatment of streptozotocin-induced diabetes mellitus in mice by intra-bone marrow bone marrow transplantation plus portal vein injection of beta cells induced from bone marrow cells.

Curative therapy for diabetes mellitus mainly involves pancreas or islet transplantation to recruit insulin-producing cells. This approach is limited, however, because of both the shortage of donor organs and allograft rejection. Intra-bone marrow bone marrow transplantation (IBM-BMT) has recently been shown to be effective in inducing donor-specific tolerance in mice and rats without the use of immunosuppressants. After induction of diabetes in 15 C3H mice with streptozotocin, the mice received both allotransplants of bone marrow cells from C57BL/6 mice by IBM-BMT and injections via the portal vein of insulin-producing cells that were induced in vitro from stem cells derived from adult C57BL/6 bone marrow. We evaluated the expression of these cells by examining the expression of not only insulin but also the crucial transcription factors insulin I and insulin II. The diabetic mice were treated with IBM-BMT and precultured insulin-producing cells. Hyperglycemia was normalized by 5 days after the treatment and remained normal for more than 45 days. This strategy might be applicable to patients with type I diabetes mellitus.

Induction of heme oxygenase-1 in renovascular hypertension is associated with inhibition of apoptosis.

The goal of this study was to characterize the impact of induction or inhibition of the heme-HO system on renal apoptosis in clipped and non-clipped kidneys from 2K1C hypertensive rats. Male Sprague-Dawley rats had a 0.25 mm silver clip placed around the left renal artery. Four groups of rats were studied: sham operated animals, 2K1C control rats, 2K1C rats received weekly injections of CoPP (5 mg/100 g body wt, administered subcutaneously), and 2K1C rats pretreated with SnMP (5 mg/ 100g body wt, administered intraperitoneally three times a week). The animals were sacrificed three weeks after surgery. We measured systolic blood pressure, plasma renin activity, non-clipped and clipped kidney HO-1 and HO-2 protein expression, HO activity, heme content, nitrotyrosine levels, and activation of selected pro- and anti-apoptotic proteins. Systolic blood pressure and plasma renin activity were significantly higher in 2K1C rats compared to sham rats. Compared to kidneys from sham animals, clipped kidneys from 2K1C rats showed a significant increase in HO-1 expression with increases in HO activity (26%), heme content (47%) and nitrotyrosine levels (49%), accompanied by an increase in caspase-3 and caspase-9 activity. In contrast, non-clipped kidneys from 2K1C rats showed no differences in HO-1 expression, HO activity, heme content, nitrotyrosine levels and caspase activity compared to sham rats. In clipped kidneys from 2K1C rats, inhibition of HO activity by SnMP augmented caspase-3 and caspase-9 activity and decreased expression of the anti-apoptotic Bcl-2 protein, while induction of HO-1 with CoPP strongly inhibited the activity of both caspases and increased the induction of Bcl-2 and Bcl-xl proteins. These findings demonstrate that the clipped kidneys responded to decreased renal perfusion pressure and increased oxidative stress by activation of the heme-HO system, which exerts antiapoptotic action via mechanisms involving decreased caspase-3 and caspase-9 activity, and increased expression of antiapoptotic molecules.

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver.

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.

Heme oxygenase-1 gene expression increases vascular relaxation and decreases inducible nitric oxide synthase in diabetic rats.

Hyperglycemia represents the main cause of complication of diabetes mellitus and oxidative stress, resulting from increased generation of reactive oxygen species (ROS), and plays a crucial role in their pathogenesis. Impairment of vascular responses in diabetic rats, as a result of an increase in superoxide (O2-), formation is a major complication in diabetes. Since heme oxygenase (HO) expression regulates the level of ROS by increasing antioxidant, such as glutathione and bilirubin, we investigated whether upregulation of HO-1 modulates the levels of iNOS and eNOS and altered vascular responses to phenylephrine (PE) and acetylcholine (Ach) in aorta and femoral arteries of diabetic (streptozotocin (STZ)-induced) rats. Our results showed that iNOS expression was increased, but HO activity was reduced, in diabetic compared to nondiabetic rats (p<0.05). Upregulation of HO-1 expression by cobalt protoporphyrin (CoPP), an inducer of HO-1 protein and activity, conferred an increase in eNOS and differentially decreased iNOS protein levels (p<0.05). Isolated aortic and femoral arteries obtained from diabetic rats exhibited contraction to PE and relaxation to Ach, which were markedly increased and decreased, respectively. However, HO-1 induction in diabetic rats normalized relaxation compared to controls. Therefore, overexpression of HO-1 may mediate an increase in eNOS and a decrease in iNOS, potentially contributing to restoration of vascular responses in diabetic rats.

Heme oxygenase-1 transduction in endothelial cells causes downregulation of monocyte chemoattractant protein-1 and of genes involved in inflammation and growth.

Heme oxygenase (HO-1) has been implicated as an anti-inflammatory gene. HO-1 overexpression, transiently and chronically, affects heme protein expression, attenuates TNF-mediated cell death, and decreases adhesion molecules. We assessed the effect of oxidant-mediated agents such as glucose and heme on 8-epi-isoprostane PGF2alpha (8-epi-PGF2alpha) and monocyte chemoattractant protein-1 (MCP-1). Glucose and heme increased both 8-epi-PGF2alpha and MCP-1. Overexpression of HO-1 decreased both 8-epi-PGF2alpha and MCP-1. To identify target genes involved in HO-1-mediated regulation of inflammation, a serial analysis of gene expression mRNA profile was performed in endothelial cells (EC) overexpressing the human HO-1 gene by transduction of a retrovirus carrying the HO-1 gene. Gene arrays (differential displays among 2400 genes) were used to identify known and novel differentially expressed genes. The levels of expression for several genes were confirmed by real time PCR in cells overexpressing the HO-1 gene. In HO-1 overexpressing cells, VEGF and the prostaglandin transporter were greatly increased while MCP-1 levels were decreased by 2.5-fold. The data from this study are relevant to understanding the mechanisms underlying the pathophysiological effects of HO-1 deficiency on endothelial cell injury and inflammation.

Quantitation of human erythroid-specific porphobilinogen deaminase mRNA by the polymerase chain reaction.

Porphobilinogen deaminase (PBG-D), the third enzyme in the heme synthetic pathway, possesses two isoforms encoded by distinct mRNAs that are the result of transcription of a single gene from two promoters through differential splicing. During erythroid differentiation, only the expression of the erythroid-specific isoform (E-PBGD) was increased. A system was developed to evaluate genetic expression of E-PBGD in samples limited in cell number and/or mRNA copy. Total RNA from human cells was reverse-transcribed and amplified by the polymerase chain reaction in the same tube with an internal standard that is an in vitro transcript of a cDNA differing from its sample counterpart by a few restriction sites and 24 bp (10%) in the target region. The primers spanned through regions where sample and standard templates were identical in sequence. Amplified templates were resolved by restriction enzyme digestion and gel electrophoresis and quantified by densitometer tracing of corresponding bands on autoradiograms. When an appropriate amount of internal standard is present in the reaction mixture, the ratio of amplified sample versus standard template is proportional to the amount of sample RNA and it is therefore possible to calculate the number of specific mRNA molecules in the original sample.

Heme metabolism and erythropoiesis in abnormal iron states: role of delta-aminolevulinic acid synthase and heme oxygenase.

Heme metabolism was examined in bone marrow and hepatic cells from iron-deficient and chronically iron-overloaded rats. Results indicate that the rate limiting enzymes delta-aminolevulinic acid synthase (ALAS) and heme oxygenase were significantly elevated in the iron-overloaded hepatic and bone marrow cells and near normal levels in cells from iron-deficient rats. Conversely, delta-aminolevulinic acid dehydrase (ALAD) was depressed in iron-overloaded cells and elevated in iron-deficient cells. Erythroid colony (CFU-E) cultures demonstrated that iron-overloaded bone marrow cells were poor hemin and CFU-E responders in vitro, whereas iron-deficient marrows grew exuberant numbers of CFU-E and responded to hemin. Succinylacetone (1 mM) inhibited ALAD activity and normal CFU-E growth, and CFU-E inhibition by succinylacetone was completely overcome by the addition of hemin. Results are discussed with respect to the regulation of hepatic and bone marrow heme metabolism in abnormal iron states and the possible role of iron in the induction of heme oxygenase in chronic iron overload.

Erythropoietin controls heme metabolic enzymes in normal human bone marrow culture.

Erythropoietin (Epo) was found to act as a concentration-dependent inducer of aminolevulinic acid (ALA) synthase and porphobilinogen (PBG) deaminase in normal human bone marrow in culture. Epo increased enzymatic activities in individual plated nucleated cells. At a low concentration of Epo, heme oxygenase activity did not change in human bone marrow erythroid progenitor cells. However, Epo at a concentration of 2 U/ml increased heme oxygenase as demonstrated by an increase in both the enzyme protein and its mRNA. In experiments with an inhibitor of heme synthesis, succinylacetone (SA), Epo failed to stimulate erythroid colony-forming unit (CFU-E) growth, but this CFU-E inhibition by SA was completely overcome by the addition of hemin. Epo nevertheless potentiated induction of ALA synthase in the presence of SA. Hemin exerted its regulatory role by negative feedback on ALA synthase in the presence of SA and Epo. Heme potentiated Epo action and resulted in the increase of human marrow erythroid progenitor cell proliferation and differentiation and a concomitant stimulation of ALA synthase and PBG deaminase. The potentiating effects of hemin on CFU-E growth were observed in human bone marrow cells cultured in media supplemented with fetal calf serum or serum-free media with interleukin 3 (IL-3). These results indicate that Epo is a potent inducer of ALA synthase and PBG deaminase in normal human bone marrow. In addition, our results may explain the mechanisms by which heme potentiates Epo or IL-3 enhancement of erythropoiesis. 1) Heme may stimulate the translation of several globin and nonglobin mRNAs, including those of ALA synthase and PBG deaminase; 2) as endogenous cellular heme synthesis reaches optimal levels, heme exerts its regulatory role on ALA synthase by negative feedback inhibition. Additionally, an increase in cellular heme may lead to an increase in its own degradation by induction of heme oxygenase.

Hemin stimulation of hemopoiesis in murine long-term bone marrow culture.

The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 microM, was added weekly to LTBMC and was found to produce a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 microM) were more effective for sustained cellularity in older cultures (10-12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 microM) was found to have a beneficial effect on the support of newly seeded cultures; however, the effect of lower hemin concentrations (0.1-1 microM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of granulocyte-macrophage colony-forming units (CFU-GM), and longevity of hemopoiesis in LTBMC was significantly increased with 0.1 microM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, whereas there was a less noticeable effect on the early erythroid progenitors (erythroid burst-forming units, BFU-E). Hemin, at 0.1 microM, increased spleen colony-forming units (CFU-S) to numbers several-fold higher than those of the control. Results suggest that hemin may produce mobilization of hemopoietic cells and committed precursors from adherent cells into suspension. Further, supplementation with hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.

Long-term bone marrow stromal and hemopoietic toxicity to AZT: protective role of heme and IL-1.

We studied the immediate and long-term effects of azidothymidine (AZT) and heme on murine hemopoietic and stromal progenitor cells in vivo and in vitro. Treatment of mice for 37 days with AZT produced anemia and leukopenia, whereas combined treatment with heme abrogated some of the toxic effects which were apparent even 2 weeks after cessation of treatment. Quantitation of spleen (CFU-S), erythroid (BFU-E) and myeloid (CFU-GM) colony formation from AZT-exposed animals revealed reductions in these progenitors, and this was partially reversed after heme treatment, especially when mice were allowed a 2-week recovery period. Long-term bone marrow cultures (LTBMC) of cells from treated groups revealed difficulty in establishing an adherent cell layer (ACL) by the first week in culture. Total cellularity, CFU-S, BFU-E and CFU-GM clonogenic potential of cultures remained depressed throughout 10 weeks of culture, whereas heme treatment overcame these depressions when AZT-exposed mice were allowed to recover for 14 days prior to culture of their cells in LTBMC. Interleukin-1 (IL-1) treatment to the same recovery group of AZT-exposed mice also resulted in an improvement of CFU-GM growth in LTBMC that was not seen in the nonrecovered group. Transplantation of cells from treated mice under the renal capsule of recipient mice revealed that AZT depressed the regeneration of osteogenic and hemopoietic cell growth within ectopic foci. These effects were reversed with heme treatment in vivo. In other experiments, heme was found to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase and to potentiate the activity of AZT triphosphate against HIV-1 reverse transcriptase. In summary, these results demonstrate that AZT inhibits the growth and development of a variety of hemopoietic, stromal and adherent cells in vivo and in vitro. Treatment of animals with heme produced recovery to near normal levels and suggests possible therapeutic potential.

Human CD34+ hematopoietic cells transduced by retrovirus-mediated interferon alpha gene maintains regeneration capacity and engraftment in NOD/SCID mice.

To achieve long-term expression of human interferon alpha-5 (IFNalpha) gene in the bone marrow (BM) hematopoietic microenvironment, replication-deficient retroviral vector LSN-IFNalpha was used to deliver the IFNalpha gene into human BM CD34+ cells. After fibronectin-facilitated transduction, a fraction of CD34+ cells was plated in methylcellulose medium with or without G418 to assess transduction efficiency and the effect of IFNalpha gene transfer on colony formation. Colony-forming assay in the presence of G418 (400 microg/mL) revealed that 41% CFU-GM colonies are G418 resistant after infection with LSN-IFNalpha retrovirus. There was no significant difference in CFU-GM/BFU-E colony formation among IFNalpha gene-transduced CD34+ cells, control vector (LXSN) transduced-CD34+ cells and nontransduced CD34+ cells. Another portion of CD34+ cells was grown in liquid medium to measure IFNalpha production. RIA revealed that IFNalpha gene-transduced CD34+ cells produced 72.2 +/- 15.4 U/mL (10(6) cells/24 hours) of IFNalpha compared with 8.3 +/- 2.1 U/mL and 4.3 +/- 1.2 U/mL in LXSN-transduced or nontransduced CD34+ cells, respectively. The remaining portion of transduced CD34+ cells was transplanted into immunodeficient (NOD/SCID) mice to allow analysis of long-term expression of IFNalpha. Transplantation of 1x10(6) CD34+ cells into sublethally irradiated NOD/SCID mice showed that IFNalpha and neo(r) mRNA were detectable in engrafted mouse BM cells for up to 6 months. We conclude that continual local expression of IFNalpha in transduced CD34+ cells does not impair either CD34+ cell growth and differentiation or engraftment and long-term survival in NOD/SCID mice.