Capillary morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis.

Anthrax protective antigen (PA) is a potent inhibitor of pathological angiogenesis with an unknown mechanism. In anthrax intoxication, PA interacts with capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Here, we show that CMG2 mediates the antiangiogenic effects of PA and is required for growth-factor-induced chemotaxis. Using specific inhibitors of CMG2 and TEM8 interaction with natural ligand, as well as mice with the CMG2 or TEM8 transmembrane and intracellular domains disrupted, we demonstrate that inhibiting CMG2, but not TEM8 reduces growth-factor-induced angiogenesis in the cornea. Furthermore, the antiangiogenic effect of PA was abolished when the CMG2, but not the TEM8, gene was disrupted. Binding experiments demonstrated a broad ligand specificity for CMG2 among extracellular matrix (ECM) proteins. Ex vivo experiments demonstrated that CMG2 (but not TEM8) is required for PA activity in human dermal microvascular endothelial cell (HMVEC-d) network formation assays. Remarkably, blocking CMG2-ligand binding with PA or CRISPR knockout abolishes endothelial cell chemotaxis but not chemokinesis in microfluidic migration assays. These effects are phenocopied by Rho inhibition. Because CMG2 mediates the chemotactic response of endothelial cells to peptide growth factors in an ECM-dependent fashion, CMG2 is well-placed to integrate growth factor and ECM signals. Thus, CMG2 targeting is a novel way to inhibit angiogenesis.

Timing of Alemtuzumab With Respect to Day of Bone Marrow Infusion and its Effects Upon Engraftment and Graft-Versus-Host Disease in Patients With Sickle Cell Disease: A Single-Institutional Study.

The possible impact of "late" alemtuzumab (administered on days -10 to -8) versus "early" alemtuzumab (-19 to -17) with respect to engraftment and acute/chronic graft-versus-host disease (GvHD) in a group of 25 pediatric patients with sickle cell disease undergoing bone marrow transplantation following conditioning with alemtuzumab, fludarabine, and melphalan is reported. The first 9 patients received "late" alemtuzumab followed by bone marrow transplantation from HLA-matched sibling donors. The next 16 patients undergoing matched sibling transplants received "early" alemtuzumab. In the "late" group, 1 patient (11%) developed acute GvHD. Six patients (67%) achieved sustained engraftment. Three patients (33%) experienced graft rejection, leading to termination of enrollment of patients on this regimen. In the "early" alemtuzumab group, acute and chronic GvHD developed in 43% and 25% patients, respectively. None of the patients experienced graft rejection in this group of patients. Three patients developed stable mixed chimerism and 13 patients demonstrated 100% donor chimerism at 1 year post-transplant and beyond. These results suggest a benefit with respect to engraftment of administering "early" versus "late" alemtuzumab in this reduced-intensity conditioning regimen, however, with the possible cost of an increase in acute, and possibly chronic GvHD.

A genomics approach to identify susceptibilities of breast cancer cells to "fever-range" hyperthermia.

BACKGROUND: Preclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to "fever range" hyperthermia (increasing the intratumoral temperature to 42-45°C) than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility. METHODS: To address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed. RESULTS: Bioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells. CONCLUSION: These data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock.

Liver transplantation for hepatitis B liver disease and concomitant hepatocellular carcinoma in the United States With hepatitis B immunoglobulin and nucleoside/nucleotide analogues.

Reinfection with hepatitis B virus (HBV) after liver transplantation (LT) may favor the recurrence of hepatocellular carcinoma (HCC), and combination therapy with hepatitis B immunoglobulin (HBIG) and nucleoside/nucleotide analogues may reduce HBV recurrence after LT. To test associations between HBV, HCC, and survival, we performed a retrospective chart review of patients undergoing LT for HBV between January 1985 and December 2010 at 7 US transplant centers. After we divided the patients into 3 eras based on evolving strategies in antiviral therapy (1985-1994, 1995-2004, and 2005-2010), we reviewed 16 variables to determine whether there were associations between survival and HCC recurrence. Seven hundred thirty-eight patients underwent transplantation for HBV, and 354 (48.0%) had concomitant HCC, which recurred in 58 patients (16.4%). Three-year survival was much better in era 3 versus era 1 (87% versus 40%, P = 0.001), and the incidence of HCC recurrence was lower (12% versus 29%, P = 0.009). The lungs were the most frequent first site of HCC recurrence, and they were followed by the liver. A multivariate analysis showed that HBV reinfection, HCC recurrence, and HBIG use were associated with worse survival (P < 0.001, P < 0.001, and P = 0.002, respectively); HCC recurrence and stage 3 HCC, among other factors, were associated with HBV reinfection (P < 0.001 and P = 0.004); and stage 3 HCC, vascular invasion of the explanted tumor, and post-LT chemotherapy were associated with HCC recurrence (P = 0.008, P < 0.001, and P < 0.001, respectively). Patients with HBV reinfection were 3.6 times more likely than patients without HBV to have HCC recurrence. These data suggest further study of attempts at LT for patients with HBV and HCC beyond the Milan criteria if their HBV is aggressively and successfully treated.

Multicenter review of liver transplant for hepatitis B-related liver disease: disparities in gender and ethnicity.

Orthotopic liver transplantation (OLT) is the preferred treatment for selected patients with hepatitis B virus (HBV)-related liver disease. This study aimed to (i) define long-term outcomes following OLT for HBV; (ii) to quantify the incidence of HBV recurrence (rHBV) as it relates to anti-HBV treatment; and (iii) to determine outcomes for specific patient subgroups. We performed a retrospective chart review of 738 patients undergoing OLT between 1985 and 2010 at seven US transplant centers and divided the patients into 3 eras, 1985-1994, 1995-2004, and 2005-2010, based on hepatitis B immunoglobulin and antiviral therapies. In Era 3, female gender (p = 0.002), recurrent hepatocellular cancer (p < 0.001), and retransplantation (p = 0.01) were significantly associated with worse survival on multivariate analysis. Survival at three yr was poor for all ethnicities in Era 1, but significantly improved for all except black Americans by Era 3. Era 2 data showed a continued increase in rHBV from five to 10 yr (16.6%, 26.2%). In conclusion, while OLT outcomes have improved because of combination antiviral and immunoglobulin therapy, women and black Americans may not have realized an equal benefit. The rate of rHBV is significant even 10 yr post-transplant with survival affected.

Prevention of de novo hepatitis B with adefovir dipivoxil in recipients of liver grafts from hepatitis B core antibody-positive donors.

Lamivudine has been shown to prevent de novo hepatitis B virus (HBV) infections in liver transplantation (LT) patients receiving hepatitis B core antibody-positive (HBcAb(+)) grafts, but it may produce long-term resistance. Adefovir dipivoxil (ADV) might be effective in preventing de novo hepatitis and resistance. A single-center, prospective trial was conducted with 16 adults (10 men and 6 women, mean age = 54 ± 11 years) who underwent LT with HBcAb(+) grafts between September 2007 and October 2009. After LT, patients were given ADV [10 mg daily (adjusted for renal function)]. No hepatitis B immune globulin was administered. At LT, all graft recipients were hepatitis B surface antigen-negative (HBsAg(-)), 38% were surface antibody-positive (HBsAb(+)), and 50% were HBcAb(+). The median follow-up after LT was 1.8 years (range = 1.0-2.6 years). All recipients had undetectable HBV DNA (<40 IU/mL) after LT until the end of follow-up. One recipient (6%) who was HBsAb(-) and HBcAb(-) before LT became HBsAg(+) after 52 weeks. One recipient was switched from ADV to entecavir for chronic renal insufficiency, and 19% of the patients had renal dose adjustments. There was a nonsignificant trend of increasing creatinine levels over time (1.2 mg/dL at LT, 1.3 mg/dL 1 year after LT, and 2.0 mg/dL 2 years after LT, P = 0.27). A comparison with a control cohort of LT recipients with hepatitis C virus who did not receive ADV showed no difference in the creatinine levels at LT or 1 year after LT. In conclusion, ADV prophylaxis prevents HBV replication in recipients of HBcAb(+) livers but does not fully protect recipients from de novo HBV. Long-term follow-up is needed to better determine the risk of de novo infection.

Distress levels in patients undergoing chemotherapy in Brazil.

OBJECTIVE: The objective of this study was to measure distress at three points during the course of chemotherapy: beginning, middle, and the last day of therapy in a private cancer center in Brazil. METHODS: One hundred patients were assessed at three points during chemotherapy using the Distress Thermometer (DT) and the Hospital Anxiety and Depression Scale (HADS). The data were analyzed with the support of SPSS 15.0 software. RESULTS: At the beginning of chemotherapy, patients mean scores were distress (82%), anxiety (78%) and depression (55%). In the middle of treatment, the percentages of distress, anxiety and depression decreased to 36.4, 25 and 25.3%, respectively. On the last day, the levels were 18.2% for distress and 14.3% for both anxiety and depression. CONCLUSION: Distress is highest in these patients at the beginning of chemotherapy, suggesting that evaluation of patients for psychosocial needs is important. Screening with a simple rapid instrument such as the DT is feasible and useful.

Severe Coronavirus Disease 2019 Infection in an Adolescent Patient After Hematopoietic Stem Cell Transplantation.

Infection with the severe acute respiratory syndrome coronavirus 2 causes severe acute lung injury in approximately 5% of infected adults, but few reports have been made of severe pediatric disease. We present an adolescent patient who contracted severe acute respiratory syndrome coronavirus 2 one week after a paternal haplo-identical hematopoietic stem cell transplant, with development of severe hyperferritinemic acute lung injury and macrophage activation-like syndrome. We present her case and a comparison of her laboratory data with those of a cohort of pediatric patients with coronavirus disease 2019 without severe disease.

Rho kinase proteins regulate global miRNA expression in endothelial cells.

BACKGROUND: Therapeutic targeting of Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) signaling for tumor cells and tumor endothelium has shown efficacy in pre-clinical tumors models, and a better understanding of how proteins regulate tumor progression will strengthen our knowledge over disease etiology and treatment of patients with cancer. Recent reports have shown that ROCK activity is critical for the expression of a large number of mRNA transcripts across multiple cell types including endothelial cells. MATERIALS AND METHODS: To examine the effects of ROCK proteins on microRNA (miRNA) expression in tumor-forming endothelial cells, we utilized microarrays to evaluate expression levels of 1088 miRNAs in vascular tumor-forming endothelial cells knocked-down for ROCK1 or ROCK2 or treated with a pharmacological inhibitor of ROCK activity. RESULTS: Microarray analysis demonstrated that inhibiting ROCK activity altered global miRNA expression. We confirmed our findings using qPCR and identified cell-cycle progression, calcium transport, and neurogenesis/synaptogenesis as the most highly overrepresented predicted target gene networks for the identified miRNAs whose expression was altered by ROCK inhibition. CONCLUSION: ROCK signaling induces large-scale changes in global miRNA expression and may lead to a better understanding of how these proteins affect aberrant vascular states.

Gene expression analysis reveals marked differences in the transcriptome of infantile hemangioma endothelial cells compared to normal dermal microvascular endothelial cells.

BACKGROUND: Infantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs). METHODS: To shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results. RESULTS: Our array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases. CONCLUSION: Our data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis of metastatic cancers.

Morphological restriction of human coronary artery endothelial cells substantially impacts global gene expression patterns.

Alterations in cell shape have been shown to modulate chromatin condensation and cell lineage specification; however, the mechanisms controlling these processes are largely unknown. Because endothelial cells experience cyclic mechanical changes from blood flow during normal physiological processes and disrupted mechanical changes as a result of abnormal blood flow, cell shape deformation and loss of polarization during coronary artery disease, we aimed to determine how morphological restriction affects global gene expression patterns. Human coronary artery endothelial cells (HCAECs) were cultured on spatially defined adhesive micropatterns, forcing them to conform to unique cellular morphologies differing in cellular polarization and angularity. We utilized pattern recognition algorithms and statistical analysis to validate the cytoskeletal pattern reproducibility and uniqueness of each micropattern, and performed microarray analysis on normal-shaped and micropatterned HCAECs to determine how constrained cellular morphology affects gene expression patterns. Analysis of the data revealed that forcing HCAECs to conform to geometrically-defined shapes significantly affects their global transcription patterns compared to nonrestricted shapes. Interestingly, gene expression patterns were altered in response to morphological restriction in general, although they were consistent regardless of the particular shape the cells conformed to. These data suggest that the ability of HCAECs to spread, although not necessarily their particular morphology, dictates their genomics patterns.

Targeting of beta adrenergic receptors results in therapeutic efficacy against models of hemangioendothelioma and angiosarcoma.

Therapeutic targeting of the beta-adrenergic receptors has recently shown remarkable efficacy in the treatment of benign vascular tumors such as infantile hemangiomas. As infantile hemangiomas are reported to express high levels of beta adrenergic receptors, we examined the expression of these receptors on more aggressive vascular tumors such as hemangioendotheliomas and angiosarcomas, revealing beta 1, 2, and 3 receptors were indeed present and therefore aggressive vascular tumors may similarly show increased susceptibility to the inhibitory effects of beta blockade. Using a panel of hemangioendothelioma and angiosarcoma cell lines, we demonstrate that beta adrenergic inhibition blocks cell proliferation and induces apoptosis in a dose dependent manner. Beta blockade is selective for vascular tumor cells over normal endothelial cells and synergistically effective when combined with standard chemotherapeutic or cytotoxic agents. We demonstrate that inhibition of beta adrenergic signaling induces large scale changes in the global gene expression patterns of vascular tumors, including alterations in the expression of established cell cycle and apoptotic regulators. Using in vivo tumor models we demonstrate that beta blockade shows remarkable efficacy as a single agent in reducing the growth of angiosarcoma tumors. In summary, these experiments demonstrate the selective cytotoxicity and tumor suppressive ability of beta adrenergic inhibition on malignant vascular tumors and have laid the groundwork for a promising treatment of angiosarcomas in humans.

Prevention of de novo hepatitis B in recipients of core antibody-positive livers with lamivudine and other nucleos(t)ides: a 12-year experience.

BACKGROUND: Lamivudine (LAM) has been shown to prevent de novo hepatitis B virus (HBV) infection in recipients of hepatitis B core antibody (HBcAb)-positive liver transplants (LT) but primarily in small studies with limited follow-up. METHODS: We conducted a retrospective cohort study of HBcAb+ graft recipients at our institution from October 1999 to August 2008. RESULTS: One hundred nineteen recipients without prior HBV were identified (median age, 54 years; 70% male), of which 62 received LAM. The median follow-up was 2.6 years overall and 5.3 years in the LAM group. Among LAM recipients, 44% were HBV naïve (HBsAb-/HBcAb-) at LT, of which 6% developed HBsAb+ and 3% developed HBcAb+ after LT. Eight percent developed de novo HBV: two recipients became hepatitis B surface antigen positive at 70 and 23 months and three experienced breakthrough with HBV DNA more than 2000 IU at 1 to 9 months after LT. Sixty percent (3 of 5) were HBV naïve. Four (6%) other recipients also had transiently detectable HBV less than 2000 IU, which did not require any changes to their prophylaxis regimen. When compared with recipients who received other nucleos(t)ide analogues, there was no difference in de novo rates: LAM 8% (5 of 62), adefovir 15% (5 of 33), tenofovir 0% (0 of 3), entecavir 0% (0 of 1), and 5% (1 of 20) for those not given prophylaxis (P=0.59). CONCLUSIONS: LAM monoprophylaxis was effective in preventing de novo HBV in the vast majority of recipients over long-term follow-up. Adefovir had a higher rate of de novo infections numerically, whereas tenofovir and entecavir had no cases and may be more effective, but this was limited by a small sample size.

Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis.

Infantile hemangiomas (IHs) are non-malignant, largely cutaneous vascular tumors affecting approximately 5-10% of children to varying degrees. During the first year of life, these tumors are strongly proliferative, reaching an average size ranging from 2 to 20 cm. These lesions subsequently stabilize, undergo a spontaneous slow involution and are fully regressed by 5 to 10 years of age. Systemic treatment of infants with the non-selective ß-adrenergic receptor blocker, propranolol, has demonstrated remarkable efficacy in reducing the size and appearance of IHs. However, the mechanism by which this occurs is largely unknown. In this study, we sought to understand the molecular mechanisms underlying the effectiveness of ß blocker treatment in IHs. Our data reveal that propranolol treatment of IH endothelial cells, as well as a panel of normal primary endothelial cells, blocks endothelial cell proliferation, migration, and formation of the actin cytoskeleton coincident with alterations in vascular endothelial growth factor receptor-2 (VEGFR-2), p38 and cofilin signaling. Moreover, propranolol induces major alterations in the protein levels of key cyclins and cyclin-dependent kinase inhibitors, and modulates global gene expression patterns with a particular affect on genes involved in lipid/sterol metabolism, cell cycle regulation, angiogenesis and ubiquitination. Interestingly, the effects of propranolol were endothelial cell-type independent, affecting the properties of IH endothelial cells at similar levels to that observed in neonatal dermal microvascular and coronary artery endothelial cells. This data suggests that while propranolol markedly inhibits hemangioma and normal endothelial cell function, its lack of endothelial cell specificity hints that the efficacy of this drug in the treatment of IHs may be more complex than simply blockage of endothelial function as previously believed.