Although present in normal cells, epidermal growth factor receptor (EGFR) is overexpressed in a variety of tumors and has been associated with decreased survival. Because activated fibroblasts are ...considered key effectors in fibrosis and because metastatic and fibrotic processes were shown to share similar signaling pathways, we investigated the contribution of EGFR signaling to idiopathic pulmonary fibrosis (IPF) progression in lung fibroblasts derived from patients with IPF (IPF-HLF). EGFR expression and EGFR-related signaling were evaluated by Western blot and immunohistochemistry. Supernatants (SN) from cultured IPF-HLF and N-HLF were added to N-HLF, and their effect on cell phenotype was tested. Growth factor levels in the SN were measured by ELISA-based arrays. EGFR activity was blocked by erlotinib (Tarceva, 0.1-0.5 µM). Expression of EGFR, phosphorylated (p)EGFR-1068 and pAkt-473 was significantly higher in IPF-HLF compared with lung fibroblasts from control donors (N-HLF) (
< 0.05). Apparent expression of p/total EGFR and pAkt-473 was found in the myofibroblastic foci of IPF patients. Erlotinib significantly inhibited IPF-HLF but not N-HLF proliferation. IPF-HLF-SN elevated N-HLF cell number, viability, EGFR expression, and pAkt-473 and ERK1/2 phosphorylation (
< 0.05). Because high basic fibroblast growth factor levels were found in the IPF-HLF-SN, nintedanib (10-100 nM) was used to inhibit fibroblast growth factor receptor (FGFR) activation. Unlike erlotinib, nintedanib completely blocked IPF-HLF-SNs' effects on the N-HLF cells in a concentration-dependent manner. In summary, IPF-HLF paracrine signaling elevates EGFR expression, which in turn, affects N-HLF survival. The FGF-EGFR interplay facilitates cellular responses that could potentially promote fibrotic disease. This interplay was successfully blocked by nintedanib.
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis. Inflammatory cytokines play a significant role in IPF pathology. However, the fibroblast itself is also ...believed to be the primary effector in IPF. We hypothesized that the fibroblasts themselves secrete pro-inflammatory cytokines that could propagate IPF by affecting normal neighboring cells. Thus, we explored the effects of IPF fibroblast derived media on normal fibroblast characteristics.
Primary IPF/normal tissue derived fibroblast cultures were established and their supernatants were collected (IPF/N-SN, respectively). These supernatants were added to normal fibroblasts. Cell death (caspase-3, western blot), proliferation, viability (WST-1), migration (scratch test) and cell detachment (crystal violet and fibronectin adhesion assays) were tested. 10 inflammatory cytokines were measured by ELISA-based quantitative array. Integrin α5 (ITGA5), pIκBα, p/total STAT3 levels were measured by western blot/IHC. TNF-α involvement was confirmed using Infliximab ®, anti-TNF-α mAb.
The IPF-SN facilitated fibroblast cell detachment and reduced cell migration (p < 0.05). Nevertheless, these effects were reversed when cells were seeded on fibronectin. The exposure to the IPF-SN also elevated ITGA5 levels, the fibronectin receptor, in addition to NFκB pathway activation (pIκBα↑ 150%, p < 0.05). In accordance, IPF derived fibroblasts were found to express higher ITGA5 than the normal cells (44%↑, p < 0.05). ITGA5 was also expressed in the fibroblastic foci. The IPF-SN contained high TNF-α levels (3-fold, p < 0.05), and Infliximab pretreatment successfully reversed all the above observations.
We suggest a possible mechanism in which IPF fibroblast secreted TNF-α modifies neighboring fibroblast cell behavior.
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The IPF-conditioned matrix (IPF-CM) system enables the study of matrix-fibroblast interplay. While effective at ...slowing fibrosis, nintedanib has limitations and the mechanism is not fully elucidated. In the current work, we explored the underlying signaling pathways and characterized nintedanib involvement in the IPF-CM fibrotic process. Results were validated using IPF patient samples and bleomycin-treated animals with/without oral and inhaled nintedanib. IPF-derived primary human lung fibroblasts (HLFs) were cultured on Matrigel and then cleared using NH
OH, creating the IPF-CM. Normal HLF-CM served as control. RNA-sequencing, PCR and western-blots were performed. HIF1α targets were evaluated by immunohistochemistry in bleomycin-treated rats with/without nintedanib and in patient samples with IPF. HLFs cultured on IPF-CM showed over-expression of 'HIF1α signaling pathway' (KEGG,
< 0.0001), with emphasis on
(PAI-1),
and
. IPF patient samples showed high HIF1α staining, especially in established fibrous tissue. PAI-1 was overexpressed, mainly in alveolar macrophages. Nintedanib completely reduced HIF1α upregulation in the IPF-CM and rat-bleomycin models. IPF-HLFs alter the extracellular matrix, thus creating a matrix that further propagates an IPF-like phenotype in normal HLFs. This pro-fibrotic loop includes the HIF1α pathway, which can be blocked by nintedanib.
Background and objective:
The term ‘fibroblast’ covers a heterogeneous cell population in idiopathic pulmonary fibrosis (IPF). The fibroblasts are considered as main effector cells, because they ...promote disease progression by releasing exaggerated amounts of extracellular matrix proteins and modifying cell microenvironment. As IPF-derived human lung fibroblasts (IPF-HLFs) were shown to express higher levels of integrin alpha-5 (ITGA5) than normal derived HLFs (N-HLFs), we explored the importance of ITGA5 to IPF progression.
Methods:
IPF-HLF and N-HLF primary cultures were established. ITGA5 was silenced by specific small interfering RNA (siRNA)s and its effects on cell phenotype (e.g. cell number, size, cell death, migration) and gene expression (e.g. RNA sequencing, quantitative polymerase chain reaction qPCR, western blot and immunofluorescence) were tested. Specific integrin expression was evaluated in IPF patient formalin-fixed paraffin embedded sections by immunohistochemistry (IHC).
Results:
ITGA5-silencing resulted in reduced IPF-HLF proliferation rates and cell migration (p < 0.05), as well as elevated cell death. transforming growth factor beta (TGF-β) targets (e.g. Fibronectin (FN1), Matrix metalloproteinase 2 (MMP2), TGFB1) were surprisingly elevated following ITGA5 silencing (p < 0.05). N-HLFs, however, were only slightly affected. Interestingly, ITGA5-silenced cells differentiated into myofibroblasts (e.g. elevated alpha-smooth muscle actin αSMA, collagen1a, large cell size). RNA-sequencing revealed that following differentiation on 3D-Matrigel for 24 h, ITGA5 levels are reduced while integrin alpha-8 (ITGA8) are elevated in IPF-HLFs. This was confirmed in IPF patients, in which ITGA5 was mainly found in fibroblastic foci, while ITGA8 was mostly observed in old fibrous tissue in the same patient.
Conclusions:
ITGA5 expression facilitates a more aggressive proliferative phenotype. Downregulation of this integrin results in myofibroblastic differentiation, which is accompanied by elevated ITGA8. Specific targeting could present a therapeutic benefit.
Background and Aims:
Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF. Galectin 3 ...(Gal-3) activates a variety of profibrotic processes. Currently, the Gal-3 inhibitor TD139 is being tested in phase II clinical trials. Since this treatment is given ‘on top’ of nintedanib, it is important to estimate its effect on Gal-3 levels. Therefore, we evaluated the impact of nintedanib on Gal-3 expression using both in vitro and in vivo models, in addition to serum samples from patients with IPF.
Methods:
Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following nintedanib treatment (10–100 nM, quantitative polymerase chain reaction), and in a silica-induced fibrosis mouse model with/without nintedanib (0.021–0.21 mg/kg) by immunohistochemistry. In addition, Gal-3 levels were analyzed in serum samples from 41 patients with interstitial lung disease patients with/without nintedanib treatment by ELISA.
Results:
Nintedanib addition to HLFs resulted in significant elevations in Gal-3, phospho-signal transducer and activator of transcription 3 (pSTAT3), as well as IL-8 mRNA levels (p < 0.05). Gal-3 expression was higher in samples from IPF patients compared with non-IPF controls at the protein and mRNA levels (p < 0.05). In the in vivo mouse model, Gal-3 levels were increased following fibrosis induction and even further increased with the addition of nintedanib, mostly in macrophages (p < 0.05). Patients receiving nintedanib presented with higher Gal-3 serum levels compared with those who did not receive nintedanib (p < 0.05).
Conclusion:
Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy for patients with IPF.
Nuclear translocation of transmembrane proteins was reported in high-grade serous ovarian cancer (HGSOC), a highly aggressive gynecological malignancy. Although the membrane receptor αvβ3 integrin is ...amply expressed in HGSOC and involved in disease progression, its nuclear localization was never demonstrated. Nuclear αvβ3 was explored in HGSOC cells (OVCAR3, KURAMOCHI, and JHOS4), nuclear localization signal (NLS) modified β3 OVCAR3, Chinese hamster ovaries (CHO-K1) and human embryonic kidney (HEK293) before/after transfections with β3/β1 integrins. We used the ImageStream technology, Western blots (WB), co immunoprecipitations (Co-IP), confocal immunofluorescence (IF) microscopy, flow cytometry for cell counts and cell cycle, wound healing assays and proteomics analyses. Fresh/archived tumor tissues were collected from nine HGSOC patients and normal ovarian and fallopian tube (FT) tissues from eight nononcological patients and assessed for nuclear αvβ3 by WB, confocal IF microscopy and immunohistochemistry (IHC). We identified nuclear αvβ3 in HGSOC cells and tissues, but not in normal ovaries and FTs. The nuclear integrin was Tyr 759 phosphorylated and functionally active. Nuclear αvβ3 enriched OVCAR3 cells demonstrated induced proliferation and oncogenic signaling, intact colony formation ability and inhibited migration. Proteomics analyses revealed a network of nuclear αvβ3-bound proteins, many of which with key cancer-relevant activities. Identification of atypical nuclear localization of the αvβ3 integrin in HGSOC challenges the prevalent conception that the setting in which this receptor exerts its pleiotropic actions is exclusively at the cell membrane. This discovery proposes αvβ3 moonlighting functions and may improve our understanding of the molecular basis of ovarian cancer pathogenesis.
To review the diagnosis and management of seven cases of Actinomyces canaliculitis.
Culture of discharge was performed in six of seven patients with Actinomyces canaliculitis using a PD Plus/F blood ...culture bottle. All patients were treated by canaliculotomy with curettage of dacryoliths, followed by treatment with systemic penicillin and Sulphacetamide drops over a period of 3-6 months. Part of the curetted concretions was fixed on a glass slide and part was sent to the laboratory for culture.
Four patients were women and three men with age ranging between 43 and 90 years. The average time lapse between onset of symptoms until diagnosis was 3 years. All cases presented with epiphora, chronic conjunctivitis, palpably thickened canaliculus, and yellow punctal discharge. Diagnosis was achieved by culture of discharge in three of six cases, culture of concretions in three of five cases, and staining of dacryoliths in all seven cases. Follow-up ranged between 12 and 48 months. The canaliculitis resolved completely and all patients have patent canalicula.
Actinomyces canaliculitis presents with epiphora, chronic purulent conjunctivitis, a palpably thickened canaliculus, and yellow punctual discharge. In suspect cases canuliculotomy and curettage should be performed, although canalicular reconstruction is generally unnecessary. Culture of discharge and concretions using PD Plus/F blood culture medium gave improved results over accepted norms. Fixation of smeared concretions on a slide in alcohol is simple and is diagnostic of the disease. We recommend long-term systemic penicillin treatment in Actinomyces canaliculitis.
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