Taimur Abbasi

Sphingolipids in Lung Endothelial Biology and Regulation of Vascular Integrity

Of the multiple and diverse homeostatic events that involve the lung vascular endothelium, participation in preserving vascular integrity and therefore organ function is paramount. We were the first to show that the lipid growth factor and angiogenic factor, sphingosine-1-phosphate, is a critical agonist involved in regulation of human lung vascular barrier function (Garcia et al. J Clin Invest, 2011). Utilizing both in vitro models and preclinical murine, rat, and canine models of acute and chronic inflammatory lung injury, we have shown that S1Ps, as well as multiple S1P analogues such as FTY720 and ftysiponate, serve as protective agents limiting the disruption of the vascular EC monolayer in the pulmonary microcirculation and attenuate parenchymal accumulation of inflammatory cells and high protein containing extravasated fluid, thereby reducing interstitial and alveolar edema. The vasculo-protective mechanism of these therapeutic effects occurs via ligation of specific G-protein-coupled receptors and an intricate interplay of S1P with other factors (such as MAPKS, ROCKs, Rho, Rac1) with rearrangement of the endothelial cytoskeleton to form strong cortical actin rings in the cell periphery and enhanced cell-to-cell and cell-to-matrix tethering dynamics. This cascade leads to reinforcement of focal adhesions and paracellular junctional complexes via cadherin, paxillin, catenins, and zona occludens. S1P through its interaction with Rac and Rho influences the cytoskeletal rearrangement indicated in the later stages of angiogenesis as a stabilizing force, preventing excessive vascular permeability. These properties translate into a therapeutic potential for acute and chronic inflammatory lung injuries. S1P has potential for providing a paradigm shift in the approach to disruption of critical endothelial gatekeeper function, loss of lung vascular integrity, and increased vascular permeability, defining features of acute lung injury (ALI), and may prove to exhibit an intrinsically protective role in the pulmonary vasculature ameliorating agonist- or sepsis-induced pulmonary injury and vascular leakage.

Hypoxic response contributes to altered gene expression and precapillary pulmonary hypertension in patients with sickle cell disease.

Background— We postulated that the hypoxic response in sickle cell disease (SCD) contributes to altered gene expression and pulmonary hypertension, a complication associated with early mortality. Methods and Results— To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 subjects with SCD with hemoglobin SS genotype and 15 subjects with Chuvash polycythemia (VHLR200W homozygotes with constitutive upregulation of hypoxia-inducible factors in the absence of anemia or hypoxia). At a 5% false discovery rate, 1040 genes exhibited >1.15-fold change in both conditions; 297 were upregulated and 743 downregulated including MAPK8 encoding a mitogen-activated protein kinase important for apoptosis, T-cell differentiation, and inflammatory responses. Association mapping with a focus on local regulatory polymorphisms in 61 patients with SCD identified expression quantitative trait loci for 103 of these hypoxia response genes. In a University of Illinois SCD cohort, the A allele of a MAPK8 expression quantitative trait locus, rs10857560, was associated with precapillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 mm Hg and pulmonary capillary wedge pressure ⩽15 mm Hg at right heart catheterization (allele frequency, 0.66; odds ratio, 13.8; n=238). This association was confirmed in an independent Walk–Treatment of Pulmonary Hypertension and Sickle Cell Disease With Sildenafil Therapy cohort (allele frequency, 0.65; odds ratio, 11.3; n=519). The homozygous AA genotype of rs10857560 was associated with decreased MAPK8 expression and present in all 14 of the identified precapillary pulmonary hypertension cases among the combined 757 patients. Conclusions— Our study demonstrates a prominent hypoxic transcription component in SCD and a MAPK8 expression quantitative trait locus associated with precapillary pulmonary hypertension.

APOL1,α-thalassemia, and BCL11A variants as a genetic risk profile for progression of chronic kidney disease in sickle cell anemia

Sickle cell anemia (SCA) is caused by a mutation in the β-globin gene that results in hemoglobin polymerization and affects approximately 1 in 500 African Americans and 25 million people worldwide. Chronic kidney disease (CKD) is observed in up to 58% of adults, and is associated with a 3-fold increased risk for early mortality in SCA. Multiple genetic modifiers of clinical complications in SCA have been reported, and combining these genetic factors into a risk profile may strengthen the predictive value as compared to the individual factors alone. Homozygosity or compound heterozygosity for APOL1 G1/G2 (G1=S342G/I384M substitutions, G2=N388/Y389 deletions) is observed in 10-15% of African Americans, and is the strongest genetic association for CKD in African Americans in general. APOL1 G1/G2 is also associated with proteinuria and albumin-

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and is a Therapeutic Target in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. Methods: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/− mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. Results: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/− mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia–induced pulmonary hypertension in rats. Conclusions: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.

Patterns of opioid use in sickle cell disease

Pain, the hallmark complication of sickle cell disease (SCD), is largely managed with opioid analgesics in the United States, but comprehensive data regarding the long‐term use of opioids in this patient population is lacking. The pain medication prescription records from a cohort of 203 SCD patients were analyzed. Twenty‐five percent were not prescribed opioid medications while 47% took only short‐acting opioids, 1% took only long‐acting opioids, and 27% took a combination of short‐acting and long‐acting opioids. The median (interquartile range) daily opioid dose was 6.1 mg (1.7–26.3 mg) of oral morphine equivalents, which is lower than the published opioid use among patients with other pain syndromes. The dose of opioids correlated with the number of admissions due to vaso‐occlusive crisis (VOC) (r = 0.53, P < 0.001). When the patients were grouped into quartiles based on daily dose opioid use, a logistic regression model showed that history of avascular necrosis (AVN) (OR: 2.87, 95% CI: 1.37–6.02, P = 0.005), 25‐OHD levels (OR: 0.59, 95% CI: 0.38–0.93, P = 0.024) and total bilirubin concentration (OR: 0.64, 95% CI: 0.42–0.99, P = 0.043) were independently associated with opioid use quartiles. In conclusion, doses and types of opioid medications used by adult SCD patients vary widely. Our findings implicate AVN and lower vitamin D levels as factors associated with higher opioid use. They also suggest an association of higher bilirubin levels, possibly suggesting higher hemolytic rate, with lower opioid use. Am. J. Hematol. 91:1102–1106, 2016.

Expression Profiling Elucidates a Molecular Gene Signature for Pulmonary Hypertension in Sarcoidosis

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

Association of circulating transcriptomic profiles with mortality in sickle cell disease

Sickle cell disease (SCD) complications are associated with increased morbidity and risk of mortality. We sought to identify a circulating transcriptomic profile predictive of these poor outcomes in SCD. Training and testing cohorts consisting of adult patients with SCD were recruited and prospectively followed. A pathway-based signature derived from grouping peripheral blood mononuclear cell transcriptomes distinguished 2 patient clusters with differences in survival in the training cohort. These findings were validated in a testing cohort in which the association between cluster 1 molecular profiling and mortality remained significant in a fully adjusted model. In a third cohort of West African children with SCD, cluster 1 differentiated SCD severity using a published scoring index. Finally, a risk score composed of assigning weights to cluster 1 profiling, along with established clinical risk factors using tricuspid regurgitation velocity, white blood cell count, history of acute chest syndrome, and hemoglobin levels, demonstrated a higher hazard ratio for mortality in both the training and testing cohorts compared with clinical risk factors or cluster 1 data alone. Circulating transcriptomic profiles are a powerful method to risk-stratify severity of disease and poor outcomes in both children and adults, respectively, with SCD and highlight potential associated molecular pathways.

Genome-Wide Analysis Identifies IL-18 and FUCA2 as Novel Genes Associated with Diastolic Function in African Americans with Sickle Cell Disease

Background Diastolic dysfunction is common in sickle cell disease (SCD), and is associated with an increased risk of mortality. However, the molecular pathogenesis underlying this development is poorly understood. The aim of this study was to identify a gene expression profile that is associated with diastolic function in SCD, potentially elucidating molecular mechanisms behind diastolic dysfunction development. Methods Diastolic function was measured via echocardiography in 65 patients with SCD from two independent study populations. Gene expression microarray data was compared with diastolic function in both study cohorts. Candidate genes that associated in both analyses were tested for validation in a murine SCD model. Lastly, genotyping array data from the replication cohort was used to derive cis-expression quantitative trait loci (cis-eQTLs) and genetic associations within the candidate gene regions. Results Transcriptome data from both patient cohorts implicated 7 genes associated with diastolic function, and mouse SCD myocardial expression validated 3 of these genes. Genetic associations and eQTLs were detected in 2 of the 3 genes, FUCA2 and IL18. Conclusions FUCA2 and IL18 are associated with diastolic function in SCD patients, and may be involved in the pathogenesis of the disease. Genetic polymorphisms within the FUCA2 and IL18 gene regions are also associated with diastolic function in SCD, likely by affecting expression levels of the genes.

Risk factors for vitamin D deficiency in sickle cell disease

Vitamin D deficiency (VDD), 25‐OHD levels <20 ng/ml, is prevalent among patients with sickle cell disease (SCD) and is linked to acute and chronic pain and bone fracture in this population. There is limited literature regarding VDD‐associated risk factors for SCD. We examined potential clinical and genomic parameters associated with VDD in 335 adults with SCD in a cross‐sectional study. VDD was present in 65% of adult SCD patients, and 25‐OHD levels independently and positively correlated with older age (P < 0·001) and vitamin D supplementation (P < 0·001). 25‐OHD levels were higher in SCD patients over 40 years of age compared to the general African‐American population. Both lower 25‐OHD levels and increased pain frequency were associated with increased expression of SLC6A5 encoding glycine transporter‐2 (GlyT2), a protein involved in neuronal pain pathways. Lower 25‐OHD levels were also associated with increased expression of CYP3A4, and with decreased expression of GC (also termed DBP) and VDR, three genes involved in vitamin D metabolism. We conclude that vitamin D supplementation should be an almost universal feature of the care of young adults with SCD, and that further research is warranted into genomic factors that regulate vitamin D metabolism in SCD.

Associations of prolonged QTc in sickle cell disease

Sudden death is a leading cause of mortality in sickle cell disease, implicating ventricular tachyarrhythmias. Prolonged QTc on an electrocardiogram (ECG), commonly seen with myocardial ischemia, is a known risk for polymorphic ventricular tachycardia (VT). We hypothesized that prolonged QTc is associated with mortality in sickle cell disease. ECG were analyzed from a cohort of 224 sickle patients (University of Illinois at Chicago, UIC) along with available laboratory, and echocardiographic findings, and from another cohort of 38 patients (University of Chicago, UC) for which cardiac MRI and free heme values were also measured. In the UIC cohort, QTc was potentially related to mortality with a hazard ratio (HR) of 1.22 per 10ms, (P = 0.015), and a HR = 3.19 (P = 0.045) for a QTc>480ms. In multivariate analyses, QTc remained significantly associated with survival after adjusting for inpatient ECG status (HR 1.26 per 10ms interval, P = 0.010) and genotype status [HR 1.21 per 10ms interval, P = 0.037). QTc trended toward association with mortality after adjusting for both LDH and hydroxyurea use (HR 1.21 per 10ms interval, P = 0.062) but was not significant after adjusting for TRV. In univariate analyses, QTc was related to markers of hemolysis including AST (P = 0.031), hemoglobin (P = 0.014), TR velocity (P = 0.036), higher in inpatients (P<0.001) and those with an SS compared to SC genotype (P<0.001) in the UIC cohort as well as to free heme in the UC cohort (P = 0.002). These findings support a relationship of prolonged QTc with hemolysis and potentially mortality in sickle cell disease.