Metagenomics indicate that public health risk may be higher from flooding following dry versus rainy periods.

Urban floodwater could lead to significant risk for public and environmental health from mobilization of microbial pathogens and overflow of wastewater treatment systems. Here, we attempted to assess this risk by obtaining metagenomic profiles of antibiotic resistance genes (ARGs), virulence factors (VFs) and pathogens present in floodwater samples collected in urban Atlanta, GA that were categorized in two distinct groups: floods that occurred after periods of drought and those after regular (seasonal) rain events. Even though no major (known) pathogens were present at the limit of detection of our sequencing effort (~3 Gbp/sample), we observed that floodwaters after drought showed a 2.5-fold higher abundance of both ARGs and VFs compared to floodwater after rainy days. These differences were mainly derived by several novel species of the Pseudomonas genus, which were more dominant in the former versus the latter samples and carried several genes to cope with osmotic stress in addition to ARGs and VFs. These results revealed that there are previously undescribed species that become mobilized after flooding events in the Southeast US urban settings and could represent an increased public health risk, especially after periods of drought, which warrants further attention.

Ethical dimensions of neglected tropical disease programming.

The global movement to control and eliminate neglected tropical diseases (NTDs) is grounded in an ethic of social justice, solidarity and health equity. NTD programmes deliver significant health benefits in socially complex environments characterized by poverty and economic disparity. We used two ethics frameworks-principlism and Upshur's public health framework-to examine ethical challenges faced by NTD programmes. They include management of serious adverse reactions associated with preventive chemotherapy, centralization of decision-making, 'opt-out' policies for school-based deworming, incomplete evidence for 'pro-poor' impact and persistent inequities in global partnerships. NTD programmes must actively address ethical challenges while pursuing global health goals.

Snow Mountain Virus recovery by synthetic human histo-blood group antigens is heavily influenced by matrix effects.

Noroviruses are known to bind to histo-blood group antigens (HBGAs) and the specific binding patterns depend on the virus genotype. However, the development of point-of-care diagnostic assays based on this binding has been challenging due to low assay sensitivity. This study utilized a well-defined stool collection from a GII.2 Snow Mountain Virus (SMV) human challenge study to investigate virus recovery from stool and emesis samples using HBGA-coated beads. SMV was recovered from H type III-coated beads for 13 stool specimens out of 27 SMV-positive specimens tested. After adjusting for non-specific binding to PEG-coated beads, the mean percent recovery by H type III-coated beads was 308.11% +/- 861.61. Recovery by H type III ligands was subject-specific and weakly correlated with stool consistency. Input virus titer was not correlated with SMV recovery. The results suggest that the generally low virus recovery we observed may be due to bead saturation or hindrance by existing glycans in the matrix that precluded the virus from being captured by the synthetic glycans. These results indicate a strong role for subject-specific and matrix effects in HBGA binding by SMV. Further investigation of the nature of this interference is needed to facilitate development of high sensitivity diagnostic assays.

Norovirus Seroprevalence among Adults in the United States: Analysis of NHANES Serum Specimens from 1999-2000 and 2003-2004.

Norovirus is the most common cause of epidemic and endemic acute gastroenteritis. However, national estimates of the infection burden are challenging. This study used a nationally representative serum bank to estimate the seroprevalence to five norovirus genotypes including three GII variants: GI.1 Norwalk, GI.4, GII.3, GII.4 US95/96, GII.4 Farmington Hills, GII.4 New Orleans, and GIV.1 in the USA population (aged 16 to 49 years). Changes in seroprevalence to the three norovirus GII.4 variants between 1999 and 2000, as well as 2003 and 2004, were measured to examine the role of population immunity in the emergence of pandemic GII.4 noroviruses. The overall population-adjusted seroprevalence to any norovirus was 90.0% (1999 to 2000) and 95.9% (2003 to 2004). Seroprevalence was highest to GI.1 Norwalk, GII.3, and the three GII.4 noroviruses. Seroprevalence to GII.4 Farmington Hills increased significantly between the 1999 and 2000, as well as the 2003 and 2004, study cycles, consistent with the emergence of this pandemic strain. Seroprevalence to GII.4 New Orleans also increased over time, but to a lesser degree. Antibodies against the GIV.1 norovirus were consistently detected (population-adjusted seroprevalence 19.1% to 25.9%), with rates increasing with age. This study confirms the high burden of norovirus infection in US adults, with most adults having multiple norovirus infections over their lifetime.

Optimized antiangiogenic reprogramming of the tumor microenvironment potentiates CD40 immunotherapy.

Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Differential Effects of Ang-2/VEGF-A Inhibiting Antibodies in Combination with Radio- or Chemotherapy in Glioma.

Antiangiogenic strategies have not shown striking antitumor activities in the majority of glioma patients so far. It is unclear which antiangiogenic combination regimen with standard therapy is most effective. Therefore, we compared anti-VEGF-A, anti-Ang2, and bispecific anti-Ang-2/VEGF-A antibody treatments, alone and in combination with radio- or temozolomide (TMZ) chemotherapy, in a malignant glioma model using multiparameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A lead to the strongest vascular changes, including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter was accompanied by the most effective chemotherapy-induced death of cancer cells and diminished tumor growth. This was most probably due to a better tumor distribution of the drug, decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes. Remarkably, all these parameters where reverted when radiotherapy was chosen as combination partner for anti-Ang-2/VEGF-A. In contrast, the best combination partner for radiotherapy was anti-VEGF-A. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that uninformed combination regimens of antiangiogenic and cytotoxic therapies should be avoided.

Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity.

Depletion of immunosuppressive tumor-associated macrophages (TAMs) or reprogramming toward a proinflammatory activation state represent different strategies to therapeutically target this abundant myeloid population. In this study, we report that inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling sensitizes TAMs to profound and rapid reprogramming in the presence of a CD40 agonist before their depletion. Despite the short-lived nature of macrophage hyperactivation, combined CSF-1R+CD40 stimulation of macrophages is sufficient to create a proinflammatory tumor milieu that reinvigorates an effective T cell response in transplanted tumors that are either responsive or insensitive to immune checkpoint blockade. The central role of macrophages in regulating preexisting immunity is substantiated by depletion experiments, transcriptome analysis of ex vivo sorted TAMs, and gene expression profiling of whole tumor lysates at an early treatment time point. This approach enabled the identification of specific combination-induced changes among the pleiotropic activation spectrum of the CD40 agonist. In patients, CD40 expression on human TAMs was detected in mesothelioma and colorectal adenocarcinoma.

Arid1a regulates response to anti-angiogenic therapy in advanced hepatocellular carcinoma.

BACKGROUND & AIMS: AT-rich interaction domain 1a (Arid1a), a component of the chromatin remodeling complex, has emerged as a tumor suppressor gene. It is frequently mutated in hepatocellular carcinoma (HCC). However, it remains unknown how Arid1a suppresses HCC development and whether Arid1a deficiency could be exploited for therapy, we aimed to explore these questions. METHODS: The expression of Arid1a in human and mouse HCCs was determined by immunohistochemical (IHC) staining. Gene expression was determined by quantitative PCR, ELISA or western blotting. Arid1a knockdown HCC cell lines were established by lentiviral-based shRNA. Tumor angiogenesis was quantified based on vessel density. The regulation of angiopoietin (Ang2) expression by Arid1a was identified by chromatin immunoprecipitation (ChIP) assay. The tumor promoting function of Arid1a loss was studied with a xenograft model in nude mice and diethylnitrosamine (DEN)-induced HCC in Arid1a conditional knockout mice. The therapeutic values of Ang2 antibody and sorafenib treatment were evaluated both in vitro and in vivo. RESULTS: We demonstrate that Arid1a deficiency, occurring in advanced human HCCs, is associated with increased vessel density. Mechanistically, loss of Arid1a causes aberrant histone H3K27ac deposition at the angiopoietin-2 (Ang2) enhancer and promoter, which eventually leads to ectopic expression of Ang2 and promotes HCC development. Ang2 blockade in Arid1a-deficient HCCs significantly reduces vessel density and tumor progression. Importantly, sorafenib treatment, which suppresses H3K27 acetylation and Ang2 expression, profoundly halts the progression of Arid1a-deficient HCCs. CONCLUSIONS: Arid1a-deficiency activates Ang2-dependent angiogenesis and promotes HCC progression. Loss of Arid1a in HCCs confers sensitivity to Ang2 blockade and sorafenib treatment. LAY SUMMARY: AT-rich interaction domain 1a (Arid1a), is a tumor suppressor gene. Arid1a-deficiency promotes Ang2-dependent angiogenesis leading to hepatocellular carcinoma progression. Arid1a-deficiency also sensitizes tumors to Ang2 blockade by sorafenib treatment.

Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade.

Pathological angiogenesis is a hallmark of cancer and a therapeutic target. Vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2; also known as ANG2) are proangiogenic cytokines that sustain tumor angiogenesis and limit antitumor immunity. We show that combined ANGPT2 and VEGFA blockade by a bispecific antibody (A2V) provided superior therapeutic benefits, as compared to the single agents, in both genetically engineered and transplant tumor models, including metastatic breast cancer (MMTV-PyMT), pancreatic neuroendocrine tumor (RIP1-Tag2), and melanoma. Mechanistically, A2V promoted vascular regression, tumor necrosis, and antigen presentation by intratumoral phagocytes. A2V also normalized the remaining blood vessels and facilitated the extravasation and perivascular accumulation of activated, interferon-γ (IFNγ)-expressing CD8+ cytotoxic T lymphocytes (CTLs). Whereas the antitumoral activity of A2V was, at least partly, CTL-dependent, perivascular T cells concurrently up-regulated the expression of the immune checkpoint ligand programmed cell death ligand 1 (PD-L1) in tumor endothelial cells. IFNγ neutralization blunted this adaptive response, and PD-1 blockade improved tumor control by A2V in different cancer models. These findings position immune cells as key effectors of antiangiogenic therapy and support the rationale for cotargeting angiogenesis and immune checkpoints in cancer therapy.

Heterogeneous perivascular cell coverage affects breast cancer metastasis and response to chemotherapy.

Angiogenesis and co-optive vascular remodeling are prerequisites of solid tumor growth. Vascular heterogeneity, notably perivascular composition, may play a critical role in determining the rate of cancer progression. The contribution of vascular pericyte heterogeneity to cancer progression and therapy response is unknown. Here, we show that angiopoietin-2 (Ang2) orchestrates pericyte heterogeneity in breast cancer with an effect on metastatic disease and response to chemotherapy. Using multispectral imaging of human breast tumor specimens, we report that perivascular composition, as defined by the ratio of PDGFRß- and desmin+ pericytes, provides information about the response to epirubicin but not paclitaxel. Using 17 distinct patient-derived breast cancer xenografts, we demonstrate a cancer cell-derived influence on stromal Ang2 production and a cancer cell-defined control over tumor vasculature and perivascular heterogeneity. The aggressive features of tumors and their distinct response to therapies may thus emerge by the cancer cell-defined engagement of distinct and heterogeneous angiogenic programs.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains.

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficacy.

PURPOSE: VEGF-A blockade has been clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. EXPERIMENTAL DESIGN: We have applied the recently developed CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab (Avastin), and the other arm recognizing Ang-2 based on LC06, an Ang-2 selective human IgG1 antibody. The potency of Ang-2-VEGF CrossMab was evaluated alone and in combination with chemotherapy using orthotopic and subcutaneous xenotransplantations, along with metastasis analysis by quantitative real-time Alu-PCR and ex vivo evaluation of vessels, hypoxia, proliferation, and apoptosis. The mechanism of action was further elucidated using Western blotting and ELISA assays. RESULTS: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient or cell line-derived human tumor xenografts, especially at later stages of tumor development. Ang-2-VEGF-A CrossMab treatment led to a strong inhibition of angiogenesis and an enhanced vessel maturation phenotype. Neoadjuvant combination with chemotherapy resulted in complete tumor regression in primary tumor-bearing Ang-2-VEGF-A CrossMab-treated mice. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiologic vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. CONCLUSION: These data establish Ang-2-VEGF-A CrossMab as a promising antitumor, antiangiogenic, and antimetastatic agent for the treatment of cancer.

A novel angiopoietin-2 selective fully human antibody with potent anti-tumoral and anti-angiogenic efficacy and superior side effect profile compared to Pan-Angiopoietin-1/-2 inhibitors.

There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.

Optical needle endoscope for safe and precise stereotactically guided biopsy sampling in neurosurgery.

Proper treatment of deep seated brain tumors requires correct histological diagnosis which unambiguously necessitates biopsy sampling. Stereotactically guided sampling of biopsies is widely used but bears the danger of incorrect sampling locations and damage to intracerebral blood vessels. Here, we present a minimally invasive contact endoscopic probe that can be inserted into the tissue inside a standard biopsy needle and allows for fluorescence detection of both tumorous tissue and intracerebral blood vessels. Outer diameter of our contact probe is smaller than 1.5 mm, field-of-view in the range of several hundred microns; the optical design allows for simultaneous detection and visualization of tissue autofluorescence and selective fluorescence signals from deep seated brain tumors and vasculature as shown on in vivo animal models. We demonstrate the tumor detection capability during stereotactic needle insertion in a clinical pilot trial. Using our probe, we expect stereotactic interventions to become safer and more precise and the technology might ultimately be used also for various other kinds of applications.

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling.

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

Bevacizumab has differential and dose-dependent effects on glioma blood vessels and tumor cells.

PURPOSE: Bevacizumab targets VEGF-A and has proved beneficial in glioma patients, improving clinical symptoms by the reduction of tumor edema. However, it remains controversial whether or not bevacizumab exerts antitumor effects in addition to (and potentially independent of) its effects on tumor vessels, and it is unknown what doses are needed to achieve this. EXPERIMENTAL DESIGN: We established a novel orthotopic glioma mouse model that allowed us to simultaneously study the kinetics of the morphologic and functional vascular changes, tumor growth, and the viability of individual tumor cells during the course of anti-VEGF therapy in the same microscopic tumor region in real-time. Three doses of bevacizumab were compared, a subclinical dose and two clinical doses (medium and high). RESULTS: Low (subclinical) doses of bevacizumab led to a significant reduction of the total vascular volume without affecting tumor cell viability or the overall tumor growth rates. Medium and high doses triggered a similar degree of vascular regression but significantly decreased tumor growth and prolonged survival. Remaining vessels revealed morphologic features of vascular normalization, reduced permeability, and an increase in blood flow velocity; the latter was dose dependent. We observed an uncoupling of the antitumoral and the antivascular effects of bevacizumab with the high dose only, which showed the potential to cause microregional glioma cell regression. In some tumor regions, pronounced glioma cell regression occurred even without vascular regression. In vitro, there was no effect of bevacizumab on glioma cell proliferation. CONCLUSIONS: Regression of glioma cells can occur independently from vascular regression, suggesting that high doses of bevacizumab have indirect anticancer cell properties in vivo.

Therapy and prophylaxis of brain metastases.

Metastases of various tumors to the brain account for the majority of brain cancers, and are associated with a poor prognosis. The most common primary sites are lung, breast, skin, kidney and colon; 10-40% of cancer patients develop brain metastases during the course of the disease. The incidence of brain metastasis appears to be rising; reasons may include better therapies for the systemic disease with longer survival of cancer patients but lower efficiency against brain metastases. In this article, we will discuss the conventional treatment with surgery, radiosurgery, radiotherapy and chemotherapy, but also new directions in the management of solid brain metastases. While general therapeutic nihilism should be avoided, it is important to recognize that the number of brain metastases, the extent of the systemic disease and also the tumor type have to be taken into account when choosing individual treatment regimens. Finally, special emphasis will be put on established and future approaches to prevent the disease. We thus aim to provide a framework for treating patients with different presentations of brain metastases, and to highlight important avenues for research.

Real-time imaging reveals the single steps of brain metastasis formation.

Brain metastasis frequently occurs in individuals with cancer and is often fatal. We used multiphoton laser scanning microscopy to image the single steps of metastasis formation in real time. Thus, it was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months. The essential steps in this model were arrest at vascular branch points, early extravasation, persistent close contacts to microvessels and perivascular growth by vessel cooption (melanoma) or early angiogenesis (lung cancer). Inefficient steps differed between the tumor types. Long-term dormancy was only observed for single perivascular cancer cells, some of which moved continuously. Vascular endothelial growth factor-A (VEGF-A) inhibition induced long-term dormancy of lung cancer micrometastases by preventing angiogenic growth to macrometastases. The ability to image the establishment of brain metastases in vivo provides new insights into their evolution and response to therapies.

Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis.

Infiltration of cancer cells into normal tissue is a hallmark of malignant gliomas and compromises treatment options. A lack of appropriate models limits the study of this invasion in vivo, which makes it difficult to fully understand its anatomy and the role of dynamic interactions with structures of the normal brain. We developed a novel methodology by utilizing multiphoton laser scanning microscopy (MPLSM) to image the movement of glioma cells deep within the normal brain of live mice in real time. This allowed us to track the invasion of individual RFP-expressing GL261 cells in relation to perfused vasculature or GFP-labeled endothelial cells repetitively over days, up to a depth of 0.5 mm. Glioma cells moved faster and more efficiently when the abluminal site of a blood vessel was utilized for invasion. Cells that invaded perivascularly were frequently found next to (a) multiple capillary structures where microvessels run parallel to each other, (b) capillary loops or glomeruloid-like bodies, and (c) dilated capillaries. Dynamic MPLSM for more than 48 h revealed that single invasive glioma cells induced intussusceptive microvascular growth and capillary loop formation, specifically at the microvascular site with which they had contact. As the main tumor grew by cooption of existing brain vessels, these peritumoral vascular changes may create a beneficial environment for glioma growth. In conclusion, our study revealed new mechanisms of peritumoral angiogenesis and invasion in gliomas, providing an explanation for their interdependence.

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.