Mohamed E. Salama

A genetic mechanism for Tibetan high-altitude adaptation

Tibetans do not exhibit increased hemoglobin concentration at high altitude. We describe a high-frequency missense mutation in the EGLN1 gene, which encodes prolyl hydroxylase 2 (PHD2), that contributes to this adaptive response. We show that a variant in EGLN1, c.[12C>G; 380G>C], contributes functionally to the Tibetan high-altitude phenotype. PHD2 triggers the degradation of hypoxia-inducible factors (HIFs), which mediate many physiological responses to hypoxia, including erythropoiesis. The PHD2 p.[Asp4Glu; Cys127Ser] variant exhibits a lower Km value for oxygen, suggesting that it promotes increased HIF degradation under hypoxic conditions. Whereas hypoxia stimulates the proliferation of wild-type erythroid progenitors, the proliferation of progenitors with the c.[12C>G; 380G>C] mutation in EGLN1 is significantly impaired under hypoxic culture conditions. We show that the c.[12C>G; 380G>C] mutation originated ∼8,000 years ago on the same haplotype previously associated with adaptation to high altitude. The c.[12C>G; 380G>C] mutation abrogates hypoxia-induced and HIF-mediated augmentation of erythropoiesis, which provides a molecular mechanism for the observed protection of Tibetans from polycythemia at high altitude.

Impact of Mechanical Unloading on Microvasculature and Associated Central Remodeling Features of the Failing Human Heart

OBJECTIVES This study investigates alterations in myocardial microvasculature, fibrosis, and hypertrophy before and after mechanical unloading of the failing human heart. BACKGROUND Recent studies demonstrated the pathophysiologic importance and significant mechanistic links among microvasculature, fibrosis, and hypertrophy during the cardiac remodeling process. The effect of left ventricular assist device (LVAD) unloading on cardiac endothelium and microvasculature is unknown, and its influence on fibrosis and hypertrophy regression to the point of atrophy is controversial. METHODS Hemodynamic data and left ventricular tissue were collected from patients with chronic heart failure at LVAD implant and explant (n = 15) and from normal donors (n = 8). New advances in digital microscopy provided a unique opportunity for comprehensive whole-field, endocardium-to-epicardium evaluation for microvascular density, fibrosis, cardiomyocyte size, and glycogen content. Ultrastructural assessment was done with electron microscopy. RESULTS Hemodynamic data revealed significant pressure unloading with LVAD. This was accompanied by a 33% increase in microvascular density (p = 0.001) and a 36% decrease in microvascular lumen area (p = 0.028). We also identified, in agreement with these findings, ultrastructural and immunohistochemical evidence of endothelial cell activation. In addition, LVAD unloading significantly increased interstitial and total collagen content without any associated structural, ultrastructural, or metabolic cardiomyocyte changes suggestive of hypertrophy regression to the point of atrophy and degeneration. CONCLUSIONS The LVAD unloading resulted in increased microvascular density accompanied by increased fibrosis and no evidence of cardiomyocyte atrophy. These new insights into the effects of LVAD unloading on microvasculature and associated key remodeling features might guide future studies of unloading-induced reverse remodeling of the failing human heart.

NEK2 Induces Drug Resistance Mainly through Activation of Efflux Drug Pumps and Is Associated with Poor Prognosis in Myeloma and Other Cancers

Using sequential gene expression profiling (GEP) samples, we defined a major functional group related to drug resistance that contains chromosomal instability (CIN) genes. One CIN gene in particular, NEK2, was highly correlated with drug resistance, rapid relapse, and poor outcome in multiple cancers. Overexpressing NEK2 in cancer cells resulted in enhanced CIN, cell proliferation and drug resistance, while targeting NEK2 by NEK2 shRNA overcame cancer cell drug resistance and induced apoptosis in vitro and in a xenograft myeloma mouse model. High expression of NEK2 induced drug resistance mainly through activation of the efflux pumps. Thus, NEK2 represents a strong predictor for drug resistance and poor prognosis in cancer and could be an important target for cancer therapy.

Identification and Acute Targeting of Gaps in Atrial Ablation Lesion Sets Using a Real-Time Magnetic Resonance Imaging System

Background— Radiofrequency ablation is routinely used to treat cardiac arrhythmias, but gaps remain in ablation lesion sets because there is no direct visualization of ablation-related changes. In this study, we acutely identify and target gaps using a real-time magnetic resonance imaging (RT-MRI) system, leading to a complete and transmural ablation in the atrium. Methods and Results— A swine model was used for these studies (n=12). Ablation lesions with a gap were created in the atrium using fluoroscopy and an electroanatomic system in the first group (n=5). The animal was then moved to a 3-tesla MRI system where high-resolution late gadolinium enhancement MRI was used to identify the gap. Using an RT-MRI catheter navigation and visualization system, the gap area was ablated in the MR scanner. In a second group (n=7), ablation lesions with varying gaps in between were created under RT-MRI guidance, and gap lengths determined using late gadolinium enhancement MR images were correlated with gap length measured from gross pathology. Gaps up to 1.0 mm were identified using gross pathology, and gaps up to 1.4 mm were identified using late gadolinium enhancement MRI. Using an RT-MRI system with active catheter navigation gaps can be targeted acutely, leading to lesion sets with no gaps. The correlation coefficient (R2) between the gap length was identified using MRI, and the gross pathology was 0.95. Conclusions— RT-MRI system can be used to identify and acutely target gaps in atrial ablation lesion sets. Acute targeting of gaps in ablation lesion sets can potentially lead to significant improvement in clinical outcomes.

Interstitial volume modulates the conduction velocity-gap junction relationship

Cardiac conduction through gap junctions is an important determinant of arrhythmia susceptibility. Yet, the relationship between degrees of G(j) uncoupling and conduction velocity (θ) remains controversial. Conflicting results in similar experiments are normally attributed to experimental differences. We hypothesized that interstitial volume modulates conduction velocity and its dependence on G(j). Interstitial volume (V(IS)) was quantified histologically from guinea pig right ventricle. Optical mapping was used to quantify conduction velocity and anisotropy (AR(θ)). Albumin (4 g/l) decreased histologically assessed V(IS), increased transverse θ by 71 ± 10%, and lowered AR(θ). Furthermore, albumin did not change isolated cell size. Conversely, mannitol increased V(IS), decreased transverse θ by 24 ± 4%, and increased AR(θ). Mannitol also decreased cell width by 12%. Furthermore, mannitol was associated with spontaneous ventricular tachycardias in three of eight animals relative to zero of 15 during control. The θ-G(j) relationship was assessed using the G(j) uncoupler carbenoxolone (CBX). Whereas 13 μM CBX did not significantly affect θ during control, it slowed transverse θ by 38 ± 9% during mannitol (edema). These data suggest changes in V(IS) modulate θ, AR(θ), and the θ-G(j) relationship and thereby alter arrhythmia susceptibility. Therefore, V(IS) may underlie arrhythmia susceptibility, particularly in diseases associated with gap junction remodeling.

Myocardial Atrophy and Chronic Mechanical Unloading of the Failing Human Heart : Implications for Cardiac Assist Device–Induced Myocardial Recovery

BACKGROUND In animal models of heterotopic transplantation, mechanical unloading of the normal, nonhypertrophic heart results in atrophy. Primarily on the basis of these animal data, the notion that chronic left ventricular assist device (LVAD)-induced unloading will result in atrophy has dominated the clinical heart failure field, and anti-atrophic drugs have been used to enhance the cardiac recovery potential observed in some LVAD patients. However, whether unloading-induced atrophy in experimental normal heart models applies to failing and hypertrophic myocardium in heart failure patients unloaded by continuous-flow LVADs has not been studied. OBJECTIVES The study examined whether mechanical unloading by continuous-flow LVAD leads to myocardial atrophy. METHODS We prospectively examined myocardial tissue and hemodynamic and echocardiographic data from 44 LVAD patients and 18 untransplanted normal donors. RESULTS Cardiomyocyte size (cross-sectional area) decreased after LVAD unloading from 1,238 ± 81 μm(2) to 1,011 ± 68 μm(2) (p = 0.001), but not beyond that of normal donor hearts (682 ± 56 μm(2)). Electron microscopy ultrastructural evaluation, cardiomyocyte glycogen content, and echocardiographic assessment of myocardial mass and left ventricular function also did not suggest myocardial atrophy. Consistent with these findings, t-tubule morphology, cytoplasmic penetration, and distance from the ryanodine receptor were not indicative of ongoing atrophic remodeling during LVAD unloading. Molecular analysis revealed no up-regulation of proatrophic genes and proteins of the ubiquitin proteasome system. CONCLUSIONS Structural, ultrastructural, microstructural, metabolic, molecular, and clinical functional data indicated that prolonged continuous-flow LVAD unloading does not induce hypertrophy regression to the point of atrophy and degeneration. These findings may be useful in designing future investigations that combine LVAD unloading and pharmaceutical therapies as a bridge to recovery of the failing heart.

Primary cutaneous mucinous carcinoma: Presence of myoepithelial cells as a clue to the cutaneous origin

Background:Primary cutaneous mucinous carcinoma (PCMC) is a rare malignancy with probable apocrine differentiation. It is important to differentiate it from metastatic mucinous carcinoma (MMC), especially from the breast. The histologic and immunohistochemical features overlap between PCMC and breast mucinous carcinomas. In this study, we introduce the presence of myoepithelial component in PCMC as a new morphologic parameter to distinguish it from MMC from either breast or sites elsewhere in the body. Materials and Methods:We studied 7 cases of PCMC. The possible in situ component in the tumor was assessed by the presence of a peripheral myoepithelial cell layer. Myoepithelial cell differentiation was confirmed with immunohistochemical stains for p63, CK 5/6, calponin, smooth muscle actin (SMA), HHF-35, and CD10. Estrogen and progesterone receptor (ER/PR), gross cystic disease fluid protein (GCDFP 15), CK7, CK20, and S-100 immunostains were also performed. Results:Histologically, multiple small monomorphic epithelial islands floating in multilocular pools of mucin characterized the tumor. Focally, epithelial islands were bordered by dermal connective tissue at the periphery of mucin pools. Secretory snouts were apparent in all cases providing evidence for apocrine differentiation. In 5 of the 7 cases, an in situ component was identified as epithelial islands being bounded by a myoepithelial layer, which was highlighted by p63, CK 5/6, calponin, SMA, and HHF-35. ER/PR and CK7 were positive in all the cases. GCDFP-15 and CD10 were focally positive in the tumor cells and myoepithelial cells, respectively. All 7 cases were negative for S-100 and CK 20. Conclusion:We conclude that an in situ component is frequently present in PCMC (5/7) and may help in distinguishing this entity from MMC, especially of breast origin. Furthermore, it may provide insight into the pathogenetic mechanism of mucinous carcinoma evolving from in situ carcinoma with luminal mucinous distention to cellular tumor with a little surrounding mucin.

Extracellular sodium and potassium levels modulate cardiac conduction in mice heterozygous null for the Connexin43 gene

Several studies have disagreed on measurements of cardiac conduction velocity (CV) in mice with a heterozygous knockout of the connexin gene Gja1—a mutation that reduces the gap junction (GJ) protein, Connexin43 (Cx43), by 50 %. We noted that perfusate ionic composition varied between studies and hypothesized that extracellular ionic concentration modulates CV dependence on GJs. CV was measured by optically mapping wild-type (WT) and heterozygous null (HZ) hearts serially perfused with solutions previously associated with no change (Solution 1) or CV slowing (Solution 2). In WT hearts, CV was similar for Solutions 1 and 2. However, consistent with the hypothesis, Solution 2 in HZ hearts slowed transverse CV (CVT) relative to Solution 1. Previously, we showed CV slowing in a manner consistent with ephaptic conduction correlated with increased perinexal inter-membrane width (WP) at GJ edges. Thus, WP was measured following perfusion with systematically adjusted [Na+]o and [K+]o in Solutions 1 and 2. A wider WP was associated with reduced CVT in WT and HZ hearts, with the greatest effect in HZ hearts. Increasing [Na+]o increased CVT only in HZ hearts. Increasing [K+]o slowed CVT in both WT and HZ hearts with large WP but only in HZ hearts with narrow WP. Conclusion: When perinexi are wide, decreasing excitability by modulating [Na+]o and [K+]o increases CV sensitivity to reduced Cx43. By contrast, CV is less sensitive to Cx43 and ion composition when perinexi are narrow. These results are consistent with cardiac conduction dependence on both GJ and non-GJ (ephaptic) mechanisms.

Bruton Tyrosine Kinase Is a Therapeutic Target in Stem-like Cells from Multiple Myeloma

Ibrutinib (Imbruvica), a small-drug inhibitor of Bruton tyrosine kinase (BTK), is currently undergoing clinical testing in patients with multiple myeloma, yet important questions on the role of BTK in myeloma biology and treatment are outstanding. Using flow-sorted side population cells from human myeloma cell lines and multiple myeloma primary samples as surrogate for the elusive multiple myeloma stem cell, we found that elevated expression of BTK in myeloma cells leads to AKT/WNT/β-catenin-dependent upregulation of key stemness genes (OCT4, SOX2, NANOG, and MYC) and enhanced self-renewal. Enforced transgenic expression of BTK in myeloma cells increased features of cancer stemness, including clonogenicity and resistance to widely used myeloma drugs, whereas inducible knockdown of BTK abolished them. Furthermore, overexpression of BTK in myeloma cells promoted tumor growth in laboratory mice and rendered side population-derived tumors that contained high levels of BTK more sensitive to the selective, second-generation BTK inhibitor, CGI1746, than side population-derived tumors that harbored low levels of BTK. Taken together, these findings implicate BTK as a positive regulator of myeloma stemness and provide additional support for the clinical testing of BTK-targeted therapies in patients with myeloma.

Optimized immunohistochemical panel to differentiate myeloid sarcoma from blastic plasmacytoid dendritic cell neoplasm

Myeloid sarcoma (MS) and blastic plasmacytoid dendritic cell neoplasm (BPDCN) can be difficult to distinguish morphologically, even with the use of extensive immunohistochemical studies. Three new research markers, myxovirus A (MxA), CLA/CD162, and CD303/BDCA-2, have been reported to be positive in BPDCN, but their clinical utility has never been tested. We compared these markers to other antibodies that have been used traditionally to distinguish MS from BPDCN to assess the utility of these newer antibodies in differential diagnosis. Formalin-fixed, paraffin-embedded tissue sections of 23 MS and 17 BPDCN cases were assessed using immunohistochemical analysis for CD4, CD14, CD33, CD43, CD56, CD68, CD123, CD163, myeloperoxidase, lysozyme, terminal deoxynucleotidyl transferase (TdT), T-cell leukemia 1 (TCL-1), MxA, cutaneous lymphocyte-associated antigen (CLA)/CD162, and blood dendritic cell antigen 2 (BDCA2)/CD303. We identified antibodies with a high predictive value of ≥90% and used these markers to develop an approach to classification using specific staining criteria. Diagnostic classification criteria were based on staining patterns of one or more of the seven markers. BPDCN was associated with positive staining for CD56, TdT, or TCL1, or negative staining for lysozyme. MS was associated with positive staining for lysozyme or myeloperoxidase, or negative staining for CD56, CD123, myxovirus, or TCL1. The immunohistochemical staining patterns observed using a panel that includes MPO, CD56, CD123, TCL1, TdT, and MxA, are predictive of MS or BPDCN. In this study, neither CD162 nor CD303 had good predictive value in distinguishing MS from BPDCN.