Evaluation of renal tubular function by multiparametric functional MRI in early diabetes.

Purpose To evaluate the tubular function in an alloxan-induced type 1 diabetes mellitus (DM) rabbit model measured by renal oxygenation (R2*), oxygen extraction fraction (OEF), and renal blood flow (RBF) using blood oxygenation level dependent, asymmetric spin echo, and arterial spin labeling MRI. Methods Twenty-six rabbits were randomized into the 3-day DM group (n = 13) and the 7-day DM group (n = 13). We performed pairs of multiparametric MRIs (before and after furosemide injection) at baseline and 3/7 days post-DM, and scored pathological kidney injury. We performed statistical analyses using non-parametric, chi-square, and Spearman correlation tests. Results At baseline, medullary R2* significantly decreased by 24.97% and 16.74% in the outer and inner stripes of the outer medulla (OS and IS, p = 0.006 and 0.003, respectively) after furosemide administration. While the corresponding OEF decreased by 15.91% for OS and 16.67% for IS (both p = 0.003), and no significant change in medullary RBF was observed (p > 0.05). In the 3-day DM group, the decrease of medullary R2* and OEF post-furosemide became unremarkable, suggesting tubular dysfunction. We noticed similar changes in the 7-day DM group. Correlation analysis showed pathological tubular injury score significantly correlated with medullary ∆R2* (post-furosemide - pre-furosemide difference, r = 0.82 for OS and 0.82 for IS) and ∆OEF (r = 0.82 for OS and 0.82 for IS) (p < 0.001, respectively). Conclusion: The combination of medullary OEF and R2* in response to furosemide could detect renal tubular dysfunction in early DM.

Quantitative measurement of the ureter on three-dimensional magnetic resonance urography images using deep learning.

BACKGROUND: Accurate measurement of ureteral diameters plays a pivotal role in diagnosing and monitoring urinary tract obstruction (UTO). While three-dimensional magnetic resonance urography (3D MRU) represents a significant advancement in imaging, the traditional manual methods for assessing ureteral diameters are characterized by labor-intensive procedures and inherent variability. In the realm of medical image analysis, deep learning has led to a paradigm shift, yet the development of a comprehensive automated tool for the precise segmentation and measurement of ureters in MR images is an unaddressed challenge. PURPOSE: The ureter was quantitatively measured on 3D MRU images using a deep learning model. METHODS: A retrospective cohort of 445 3D MRU scans (443 patients, 52 ± 18 years; 217 female patients) was collected and split into training, validation, and internal testing cohorts. A 3D V-Net model was trained for urinary tract segmentation, and a post-processing algorithm was developed for ureteral measurements. The accuracy of the segmentation was evaluated using the Dice similarity coefficient (DSC) and volume intraclass correlation coefficient (ICC), with ground truth segmentations provided by experienced radiologists. The external cohort comprised 50 scans (50 patients, 55 ± 21 years; 30 female patients), and the model-predicted ureteral diameter measurements were compared with manual measurements to assess system performance. The various diameter parameters of ureter among the different measurement methods (ground truth, auto-segmentation with automatic diameter extraction, and manual segmentation with automatic diameter extraction) were assessed with Friedman tests and post hoc Dunn test. The effectiveness of the UTO diagnosis was assessed by receiver operating characteristic (ROC) curves and their respective areas under the curve (AUC) between different methods. RESULTS: In both the internal test and external cohorts, the mean DSC values for bilateral ureters exceeded 0.70. The ICCs for the bilateral ureter volume obtained by comparing the model and manual segmentation were all greater than 0.96 (p  < â€¯0.05), except for the right ureter in the internal test cohort, for which the ICC was 0.773 (p  < â€¯0.05). The mean DSCs for interobserver and intraobserver reliability were all above 0.97. The maximum diameter of the ureter exhibited no statistically significant differences either in the dilated (p = 0.08) or in the non-dilated (p = 0.32) ureters across the three measurement methods. The AUCs of ground truth, auto-segmentation with automatic diameter extraction, and manual segmentation with automatic diameter extraction in diagnosing UTO were 0.988 (95% CI: 0.934, 1.000), 0.961 (95% CI: 0.893, 0.991), and 0.979 (95% CI: 0.919, 0.998), respectively. There was no statistical difference between AUCs of the different methods (p > 0.05). CONCLUSION: The proposed deep learning model and post-processing algorithm provide an effective means for the quantitative evaluation of urinary diseases using 3D MRU images.

Characteristics of Sciatic Scoliotic List in Lumbar Disc Herniation.

STUDY DESIGN: Retrospective radiological study. OBJECTIVES: Physical examination reveals sciatic scoliotic list (SSL) in some patients with lumbar disc herniation (LDH). We aimed to evaluate the coronal and sagittal parameters of the SSL curve, describe their characteristics, and establish a new classification system. METHODS: We retrospectively reviewed 230 patients (SSL group = 96; non-SSL group = 134) diagnosed with single-segment LDH from January 2018 to December 2020. The control group comprised 70 healthy volunteers. Radiographic parameters were compared between the groups. RESULTS: In the SSL group, the Cobb's angle was 12.5 ± 5.3° (4.2-31.2), trunk shift 26.2 ± 17.9 mm (.0-88.2 mm), and apical vertebral translation 31.7 ± 16.0 mm (1.18-8.58 mm). Further, 62.5% of patients had L4/5 disc herniation, 76.0% had disc herniation at the convex side of the lumbosacral scoliosis, and 81.3% had disc herniation at the opposite side of the trunk shift. Compared to the control group, lumbar lordosis and thoracic kyphosis decreased, pelvic tilt increased, and the sagittal vertical axis moved forward in the other patients. The sagittal imbalance in the SSL group exacerbated. Using the positional relationship between vertebrae and the central sacral vertical line (CSVL), we identified two main SSL patterns with which 95.8% of the patients were defined as Type 1. CONCLUSIONS: The SSL curve is characterized by a long thoracic or thoracolumbar curve, with a relatively straight sagittal profile and little rotation. Further, the lumbar and thoracic vertebrae shifts are on the same side as the CSVL. These features of the SSL curve differentiate it from other types of structural scoliosis.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Proteomic analysis reveals the mechanism of different environmental stress-induced tolerance of Pseudomonas aeruginosa to monochloramine disinfection.

Although drinking water disinfection proved to be an effective strategy to eliminate many pathogens, bacteria can still show disinfection tolerance in drinking water distribution systems. To date, the molecular mechanisms on how environmental stress affects the tolerance of Pseudomonas aeruginosa to monochloramine are not well understood. Here, we investigated how three stress conditions, namely starvation, low temperature, and starvation combined with low temperature, affected the monochloramine tolerance of Pseudomonas aeruginosa, an opportunistic pathogen commonly found in drinking water distribution systems. All stress conditions significantly promoted monochloramine tolerance, among which starvation had the most drastic effects. Proteomic analyses suggested that the three conditions not only triggered a positive antioxidant defense against oxidative damages but also prepared the bacteria to employ a passive defense mechanism against disinfectants via dormancy. Moreover, the expression of antioxidant enzymes reached the maximum under the starvation condition and further low temperature treatment had little effect on bacterial response to oxidative stress. Instead, we found further treatment of the starved cells with low temperature decreased the osmotic stress response and the stringent response, which generally play pivotal roles in disinfection tolerance. Taken together, these findings shed light on how abiotic factors influence the bacterial disinfection tolerance and will aid design of efficient strategies to eliminate Pseudomonas aeruginosa from drinking water.

Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

Bacterial type II toxin-antitoxin systems acting through post-translational modifications.

The post-translational modification (PTM) serves as an important molecular switch mechanism to modulate diverse biological functions in response to specific cues. Though more commonly found in eukaryotic cells, many PTMs have been identified and characterized in bacteria over the past decade, highlighting the importance of PTMs in regulating bacterial physiology. Several bacterial PTM enzymes have been characterized to function as the toxin component of type II TA systems, which consist of a toxin that inhibits cell growth and an antitoxin that protects the cell from poisoning by the toxin. While TA systems can be classified into seven types based on nature of the antitoxin and its activity, type II TA systems are perhaps the most studied among the different TA types and widely distributed in eubacteria and archaea. The type II toxins possessing PTM activities typically modify various cellular targets mostly associated with protein translation and DNA replication. This review mainly focuses on the enzymatic activities, target specificities, antitoxin neutralizing mechanisms of the different families of PTM toxins. We also proposed that TA systems can be conceptually viewed as molecular switches where the 'on' and 'off' state of the system is tightly controlled by antitoxins and discussed the perspective on toxins having other physiologically roles apart from growth inhibition by acting on the nonessential cellular targets.

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice.

Hydrogen sulfide (H(2)S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H(2)S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H(2)S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine ß-synthase (CBS) has been shown to catalyze H(2)S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H(2)S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H(2)S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H(2)S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H(2)S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H(2)S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H(2)S for the treatment of cardiovascular and other diseases.

Suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo.

Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had approximately 80% lower hepatic PC-1 mRNA levels, approximately 30% lower ambient fed plasma glucose, approximately 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes.

Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes.

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.

D-4F induces heme oxygenase-1 and extracellular superoxide dismutase, decreases endothelial cell sloughing, and improves vascular reactivity in rat model of diabetes.

BACKGROUND: Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals. METHODS AND RESULTS: We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 microg/100 g of body weight, intraperitoneal injection) on superoxide (O2-), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2- levels were elevated in diabetic rats (74.8+/-8x10(3) cpm/10 mg protein) compared with controls (38.1+/-8x10(3) cpm/10 mg protein; P<0.01). D-4F decreased O2- levels to 13.23+/-1x10(3) (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50+/-6 cells/mL) than in controls (5+/-1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20+/-3 cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0+/-2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level. CONCLUSIONS: We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.

Angiotensin II induces carbon monoxide production in the perfused kidney: relationship to protein kinase C activation.

Heme oxygenase (HO)-derived carbon monoxide (CO) attenuates vascular reactivity to constrictor stimuli. ANG II produces vasoconstriction and induces HO-1 isoform expression. However, direct evidence that ANG II promotes HO product generation is lacking. Therefore, we examined the effects of ANG II on CO release and HO isoform expression in isolated rat kidneys. Kidneys were perfused with oxygenated Krebs buffer. ANG II (1 micromol/l) increased (P < 0.05) perfusion pressure from 97 +/- 9 to 150 +/- 14 mmHg; it also increased (P < 0.05) the concentration of CO in the venous effluent (from 27.1 +/- 11.9 to 45.6 +/- 11.7, 62.5 +/- 16.7, 94.8 +/- 20.7, and 101.9 +/- 13.1 nmol/l after 30, 60, 90, and 120 min, respectively). The pressor effect of ANG II was blunted (P < 0.05) in kidneys perfused with buffer containing losartan (10 micromol/l) or PKC inhibitors staurosporine (0.1 micromol/l) or calphostin C (1 micromol/l). Kidneys perfused with buffer containing ANG II for 120 min also displayed increased (P < 0.05) HO-1 expression. Stannous mesoporphyrin (30 micromol/l) decreased CO release (P < 0.05) in preparations perfused with and without ANG II; the HO inhibitor also increased (P < 0.05) perfusion pressure, more so in kidneys perfused with that without ANG II. We conclude that ANG II stimulates CO production and release in isolated, perfused rat kidneys. This action of ANG II is linked to the activation of AT(1) receptors and involves PKC activation and upregulation of renal HO-1 but not of HO-2 protein expression. The study suggests upregulation of renal HO-1 and CO release are protagonic events in a counterregulatory mechanism that attenuates ANG II-induced renal vasoconstriction.

Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (P<0.05) in LXSN rats (23+/-1, 37+/-2, and 52+/-2 at 0.5, 2, and 10 ng). Importantly, treating LSN-HHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli.

Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin II-mediated increase in oxidative injury.

BACKGROUND: Heme oxygenase-1 (HO-1) catalyzes the conversion of heme to bilirubin, carbon monoxide (CO), and free iron, thus controlling the level of cellular heme. The medullary thick ascending limb of the loop of Henle (TALH) is situated in a site of markedly diminished oxygen tension and, as such, is highly vulnerable to ischemic insult. We hypothesize that selective upregulation of HO-1 in TALH by gene transfer attenuates oxidative stress caused by angiotensin II (Ang II). METHODS: An adenoviral vector expressing the human HO-1 under the control of the TALH-specific promoter [Na(+)-K(+)-Cl(-) cotransporter (NKCC2 promoter)] was constructed and the cell specific expression of the recombinant adenovirus was examined using several types of cells, including endothelial, vascular smooth muscle, and TALH cells. The effects of HO-1 transduction on HO-1 expression, HO activity and the response to Ang II with respect to cyclooxygenase-2 (COX-2) up-regulation and oxidative injury [growth-stimulating hormone (GSH) levels and cell death] were determined. RESULTS: Western blot and reverse transcription-polymerase chain reaction (RT-PCR) revealed that human HO-1 was selectively expressed in primary cultured TALH cells following infection with Ad-NKCC2-HO-1. In TALH cells infected with Ad-NKCC2-HO-1, Ang II-stimulated prostaglandin E(2) (PGE(2)) levels were reduced by 40%. Ang II caused a marked decrease in GSH levels and this decrease was greatly attenuated in TALH cells transduced with Ad-NKCC2-HO-1. Moreover, Ang II-mediated DNA degradation was completely blocked by the site-specific expression of human HO-1 gene. CONCLUSION: These results indicate that TALH cell survival after exposure to oxidative stress injury may be facilitated by selective upregulation of HO-1, thusly blocking inflammation and apoptosis.

CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin.

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 +/- 0.06 mN/mm) in pulmonary vascular rings treated with 15 micromol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 micromol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 micromol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 micromol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.

Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells.

Heme oxygenase-1 (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as heme and inflammatory molecules. Incubation of endothelial cells in a high-glucose (33 mmol/L) medium for 7 days resulted in a decrease of HO activity by 34% and a decrease in HO-1 and HO-2 proteins compared with cells exposed to low glucose (5 mmol/L) (P<0.05) or cells exposed to mannitol (33 mmol/L). Overexpression of HO-1 was coupled with an increase in HO activity and carbon monoxide synthesis, decreased cellular heme, and acceleration in all phases of the cell cycle (P<0.001). The rate of cell cycle or cell birth rate was increased by 29% (P<0.05) in cells overexpressing HO-1 but decreased by 23% (P<0.05) in cells underexpressing HO-1 compared with control cells. Exposure to high glucose significantly decreased cell-cycle progression in control cells and in cells underexpressing HO-1 but did not decrease cell-cycle progression in cells overexpressing HO-1. High glucose induced p21 and p27 in control cells but not in cells overexpressing HO-1. The addition of tin-mesoporphyrin (SnMP), an inhibitor of HO activity, reversed the HO-1-mediated decrease of p21 and p27 in cells overexpressing HO-1. These findings identify a novel effect of HO-1 on endothelial cell growth and indicate that heme metabolism and HO-1 expression regulate signaling systems in cells exposed to high glucose, which controls cell-cycle progression.

Diminished heme oxygenase potentiates cell death: pyrrolidinedithiocarbamate mediates oxidative stress.

Pyrrolidinedithiocarbamate (PDTC) is a metal-chelating compound that exerts both pro-oxidant and antioxidant effects and is widely used as an antitumor and anti-inflammatory agent. Heme oxygenase-1 (HO-1) is a redox-sensitive-inducible protein that provides efficient cytoprotection against oxidative stress. Because it has been reported that several angiogenic stimulating factors upregulating HO-1 in endothelial cells cause a significant increase in angiogenesis, we investigated the effect of PDTC on cell proliferation and angiogenesis and the effect of overexpression and underexpression of HO-1. The evaluation of PDTC (20 or 50 micro M) in endothelial cells resulted in significant increase in HO-1 mRNA and protein (P < 0.001), but a decrease in cell proliferation. Pretreatment of endothelial cells with SnCl(2) (10 micro M), an inducer of HO-1 attenuated the PDTC-mediated decrease in cell proliferation (P < 0.05). In contrast, pretreatment with SnMP, an inhibitor of HO activity, magnified the inhibiting effect of PDTC on cell proliferation. Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated the PDTC-induced decrease in cell proliferation. Underexpression of HO-1, by delivery of the human HO-1 in antisense orientation, enhanced the PDTC-mediated decrease in cell proliferation. The decrease, by PDTC, in proliferation of cells underexpressing HO-1 is related to an increase in O(-)(2) production. Collectively, these results demonstrate that upregulation of HO-1 was able to attenuate the PDTC-mediated cell proliferation, but was unable to reverse the high concentration of PDTC-induced decrease in angiogenesis.

Influence of heme and heme oxygenase-1 transfection of pulmonary microvascular endothelium on oxidant generation and cGMP.

Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells.

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.