Damien Ambrosetti

Adult Duct-Lining Cells Can Reprogram into β-like Cells Able to Counter Repeated Cycles of Toxin-Induced Diabetes

It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing β-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether α cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into β-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated α-to-β-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a β-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole β cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon+ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

Differences in visible light‐induced pigmentation according to wavelengths: a clinical and histological study in comparison with UVB exposure

The visible light spectrum is wide, and it can be hypothesized that all the wavelengths between 400–700 nm do not induce the same photobiological effects on pigmentation. We assessed the potential pro‐pigmenting effects of two single wavelengths located at both extremities of the visible spectrum: the blue/violet line (λ = 415 nm) and the red line (λ = 630 nm). We made colorimetric, clinical, and histological assessments with increasing doses of those lights on healthy volunteers. Then, we compared these irradiations to non‐exposed and UVB‐exposed skin. Colorimetric and clinical assessments showed a clear dose effect with the 415‐nm irradiation, in both skin type III and IV subjects, whereas the 630 nm did not induce hyperpigmentation. When compared to UVB irradiation, the blue–violet light induced a significantly more pronounced hyperpigmentation that lasted up to 3 months. Histological examination showed a significant increase of keratinocyte necrosis and p53 with UVB, as compared to 415‐ and 630‐nm exposures.

The CXCL7/CXCR1/2 Axis Is a Key Driver in the Growth of Clear Cell Renal Cell Carcinoma

Mutations in the von Hippel-Lindau gene upregulate expression of the central angiogenic factor VEGF, which drives abnormal angiogenesis in clear cell renal cell carcinomas (ccRCC). However, the overexpression of VEGF in these tumors was not found to correlate with overall survival. Here, we show that the proangiogenic, proinflammatory cytokine CXCL7 is an independent prognostic factor for overall survival in this setting. CXCL7 antibodies strongly reduced the growth of ccRCC tumors in nude mice. Conversely, conditional overexpression of CXCL7 accelerated ccRCC development. CXCL7 promoted cell proliferation in vivo and in vitro, in which expression of CXCL7 was induced by the central proinflammatory cytokine interleukin (IL)-1β. ccRCC cells normally secrete low amounts of CXCL7; it was more highly expressed in tumors due to high levels of IL-1β there. We found that a pharmacological inhibitor of the CXCL7 receptors CXCR1 and CXCR2 (SB225002) was sufficient to inhibit endothelial cell proliferation and ccRCC growth. Because CXCR1 and CXCR2 are present on both endothelial and ccRCC cells, their inhibition affected both the tumor vasculature and the proliferation of tumor cells. Our results highlight the CXCL7/CXCR1/CXCR2 axis as a pertinent target for the treatment of ccRCC.

EGFR mutation status in brain metastases of non-small cell lung carcinoma

Brain metastases are a frequent and grave complication of non-small cell lung carcinoma (NSCLC). The prognosis is generally poor, despite standard therapy based on surgery and radiotherapy. A degree of understanding of the molecular basis of tumors has led to the development of targeted agents with promising initial findings for the treatment of NSCLC. EGFR mutations have been identified which are associated with significant sensitivity to EGFR tyrosine kinase inhibitors (TKI) and correlate with improved outcome in patients with NSCLC who are treated with these agents. The adoption of treatment tailored to the genetic make-up of individual tumors could lead to substantial therapeutic improvements, and such targeted therapy might be considered as a therapeutic option for brain metastases in the future. We review current knowledge about EGFR mutation status in the specific context of brain metastasis: its association with the response of brain metastases to TKI, its prevalence in brain metastases, and the correlation between mutation status in metastases as compared to the corresponding primary lung carcinoma.

Tumour-derived SPARC drives vascular permeability and extravasation through endothelial VCAM1 signalling to promote metastasis

Disruption of the endothelial barrier by tumour-derived secreted factors is a critical step in cancer cell extravasation and metastasis. Here, by comparative proteomic analysis of melanoma secretomes, we identify the matricellular protein SPARC as a novel tumour-derived vascular permeability factor. SPARC deficiency abrogates tumour-initiated permeability of lung capillaries and prevents extravasation, whereas SPARC overexpression enhances vascular leakiness, extravasation and lung metastasis. SPARC-induced paracellular permeability is dependent on the endothelial VCAM1 receptor and p38 MAPK signalling. Blocking VCAM1 impedes melanoma-induced endothelial permeability and extravasation. The clinical relevance of our findings is highlighted by high levels of SPARC detected in tumour from human pulmonary melanoma lesions. Our study establishes tumour-produced SPARC and VCAM1 as regulators of cancer extravasation, revealing a novel targetable interaction for prevention of metastasis.

Prediction of membranous nephropathy recurrence after transplantation by monitoring of anti-PLA2R1 (M-type phospholipase A2 receptor) autoantibodies: a case series of 15 patients

BACKGROUND The predictive value of anti-M-type phospholipase A2 receptor (PLA2R1) autoantibodies for membranous nephropathy (MN) recurrence after renal transplantation remains controversial. METHODS Our aim was to monitor anti-PLA2R1 IgG4 activity using a sensitive enzyme-linked immunosorbent assay in 15 kidney transplant recipients with MN, and to test the correlation between antibody titres and MN recurrence. RESULTS Five patients never exhibited anti-PLA2R1 antibodies, and one of them relapsed. Ten patients (67%) had IgG4 anti-PLA2R1 antibodies at the time of transplantation and during follow-up. The presence of IgG4 anti-PLA2R1 antibodies at the time of kidney transplantation does not imply MN recurrence (P = 0.600, n = 15). However, a positive IgG4 anti-PLA2R1 activity during follow-up (>Month 6) was a significant risk factor for MN relapse (P = 0.0048, n = 10). Indeed, four patients had persistent IgG4 anti-PLA2R1 activity after transplantation and relapsed. Among them, one was successfully treated with rituximab. Another had persistently high IgG4 anti-PLA2R1 activity and exhibited a histological relapse but no proteinuria while on treatment with renin-angiotensin system inhibitors. In contrast, the six other patients who did not relapse exhibited a decrease of their IgG4 anti-PLA2R1 activity following transplant immunosuppression, including two with proteinuria due to biopsy-proven differential diagnoses. A weak transplant immunosuppressive regimen was also a risk factor of MN recurrence (P = 0.0048, n = 10). Indeed, the six patients who received both an induction therapy and a combined treatment with calcineurin inhibitors/mycophenolate exhibited a decrease of IgG4 anti-PLA2R1 activity and did not relapse, while the four patients who did not receive this strong immunosuppressive treatment association had persistently high IgG4 anti-PLA2R1 activity and relapsed. CONCLUSION The monitoring of IgG4 anti-PLA2R1 titres during follow-up helps to predict MN recurrence, and a strong immunosuppressive treatment of anti-PLA2R1 positive patients may prevent recurrence.

Bevacizumab/docetaxel association is more efficient than docetaxel alone in reducing breast and prostate cancer cell growth: A new paradigm for understanding the therapeutic effect of combined treatment

Bevacizumab (Bvz), a Vascular Endothelial Growth Factor (VEGF)-targeted humanised monoclonal antibody, provides clinical benefit in combination with docetaxel (DXL), a microtubule-stabilising agent, in the treatment of metastatic breast and prostate cancers. Since VEGF and their receptors are expressed by tumour cells, we hypothesised that Bvz, in addition to its impact on neo-vascularisation, could have an impact on tumour cells and enhance the DXL activity. Hence, we studied the effect of DXL and Bvz on metastatic breast (MDA MB-231) and prostate (PC3) cancer cells lines. Bvz alone did not decrease cell proliferation but in combination with DXL, Bvz enhanced the anti-proliferative activity of DXL. Other anti-angiogenic factors Sunitinib, Sorafenib and Gefitinib enhanced the anti-proliferative effect of DXL. qPCR experiments showed that DXL significantly increased the VEGF and VEGF receptor 2 (VEGF-R2) mRNA levels. Activation of VEGF and VEGF-R2 promoters demonstrated that enhanced mRNA levels are partly due to transcriptional activation. ELISA assays showed that DXL induced accumulation of cytoplasmic VEGF but decreased extracellular levels by 39% (MDA) and 48% (PC3). Cell surface localisation of VEGF-R2 was increased by DXL alone, but decreased after combined treatment of DXL plus Bvz. Abnormal expression of VEGF-R2 was also shown on breast and prostate tumour samples reinforcing the results obtained on cellular models. In conclusion, DXL and Bvz in combination decreased extracellular VEGF and VEGF-R2 levels at the plasma membrane thereby blocking an important growth/survival loop. Thus, the combined therapeutic impact of Bvz and DXL observed in clinical trials is associated with enhanced anti-proliferative activity and inhibition of the vascular network.

Acute kidney injury following crizotinib administration for non-small-cell lung carcinoma

A case of locally advanced non-small-cell lung carcinoma (NSCLC) was inadequately controlled with cisplatin, bevacizumab and pemetrexed chemotherapy. Following identification of a mutation of the echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) gene, crizotinib was then administered as targeted treatment. Kidney function was normal at diagnosis and during the first line therapy. The administration of crizotinib coincided on two occasions with a conspicuous rise in the serum creatinine level. Urinary protein over creatinine ratio was 0.31 g/g with 22% albumin and macroscopic hematuria. A kidney biopsy was performed at the time of the second episode of renal impairment which showed acute tubular necrosis (ATN) indicating recent renal injury together with a mononuclear cell infiltrate consistent with ongoing repair related to a previous insult. The renal lesions were closely related temporally to crizotinib administration, supporting a causative role for crizotinib in the acute renal injury and this phenomenon has not previously been described.

Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation

Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma.