EGFR and EGFR ligands in serum in healthy women; reference intervals and age dependency.

Background The epidermal growth factor receptor (EGFR) system is involved in cancer pathogenesis and serves as an important target for multiple cancer treatments. EGFR and its ligands epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), betacellulin (BTC), amphiregulin (AREG) and transforming growth factor α (TGF-α) have potential applications as prognostic or predictive serological biomarkers in cancer. The aim was to establish EGFR and EGFR ligand reference intervals in healthy women. Methods EGFR and EGFR ligands were measured in serum from 419 healthy women aged 26-78 years. The need for age partitioned reference intervals was evaluated using Lahti's method. EGFR and EGF were analyzed using ELISA assays, whereas HB-EGF, BTC, AREG and TGF-α were analyzed using the highly sensitive automated single molecule array (Simoa) enabling detection below the lower reference limit for all six biomarkers. Results Reference intervals for EGFR and the EGFR ligands were determined as the 2.5th and 97.5th percentiles. All six biomarkers were detectable in all serum samples. For EGFR, EGF, HB-EGF and TGF-α, reference intervals were established for women <55 years and for women >55 years, whilst common reference intervals were established for AREG and BTC including women aged 26-78 years. Conclusions Age specific reference intervals were determined for EGFR, EGF, HB-EGF, BTC, AREG and TGF-α.

Expression of PGR, HBEGF, ITGAV, ITGB3 and SPP1 genes in eutopic endometrium of infertile women with endometriosis during the implantation window: a pilot study.

OBJECTIVE: Alterations in endometrial receptivity may be involved in the etiopathogenesis of endometriosis-related infertility. The literature has suggested that patients with endometriosis present progestin resistance, which could affect embryo implantation. We question the presence of alterations in the expression of the progesterone receptor gene (PGR) and the genes related to endometrium-embryo interaction regulated by progesterone. This pilot study compared the expression of PGR, HBEGF, ITGAV, ITGB3, and SPP1 genes in eutopic endometrium during the implantation window (IW) in infertile women with endometriosis with that observed in the endometrium of fertile and infertile controls. METHODS: In this prospective case-control study, endometrial biopsies were performed during the IW in patients aged between 18 and 45 years old, with regular cycles and without endocrine/systemic dysfunctions, divided into endometriosis (END), infertile control (IC) and fertile control (FC) groups. Total RNA extraction, cDNA synthesis, and gene expression analysis by Real-Time PCR were performed. We assessed the size of the difference that our series was powered to detect. RESULTS: From the 687 patients who underwent diagnostic videolaparoscopy or tubal ligation at the University Hospital, 130 were eligible. Of these, 32 had endometrial samples collected, with 17 confirmed in the IW. Fifteen samples (5 END, 5 IC and 5 FC) were analyzed. There was no significant difference in the expression of any studied gene. Our sample size allowed us to identify or discard large differences (two standard deviations) among the groups. CONCLUSION: Endometriosis doesn't cause large changes in the endometrial expression of PGR, HBEGF, ITGAV, ITGB3 and SPP1 during the IW.

Serous carcinomatous component championed by heparin-binding EGF-like growth factor (HB-EGF) predisposing to metastasis and recurrence in stage I uterine malignant mixed mullerian tumor.

The stage I uterine malignant mixed mullerian tumor (MMMT) shows different potential for progression. We reason that MMMTs with high-grade carcinomatous component and positivity for HB-EGF are prone to recurrence/metastasis in the early stage. A retrospective clinical and histopathologic review with immunohistochemical staining for HB-EGF, EGFR, and integrin-α5 was performed for 62 surgically staged MMMT cases. Recurrence/metastasis (RM) is 6/18 (33%) in stage I disease. Of all the clinicopathologic variables and biomarkers analyzed for stage I MMMT, serous carcinomatous component (83% [5/6] versus 17% [1/12], P = .0015) and HB-EGF expression (100% [6/6] versus 50% [6/12], P=.0339) were significantly different between groups with RM and without RM. The presence of serous carcinoma in all stages was 83% (5/6) in stage I with RM, 8% (1/12) in stage I without RM, 20% (1/5) in stage II, 36.4% (8/22) in stage III and 64.7% (11/17) in stage IV; this was paralleled by HB-EGF expression of 100% (6/6), 50% (6/12), 40% (2/5), 50% (11/22) and 71% (12/17) with a correlation coefficient r=0.9131 (P=.027). HB-EGF and integrin-α5 were highly expressed in MMMTs bearing serous carcinoma component, compared to endometrioid and unclassifiable/miscellaneous subtypes (84.6%/47.6%/33.3%, P=.025 for HB-EGF; and 61.5%/42.9%/20.0%, P=.021 for integrin-α5). The EGFR positivity was comparable among the three subtypes (48.1%, 47.6% and 26.7%, P=.326). This study indicates that serous carcinomatous component championed by expression of HB-EGF predisposes to recurrence/metastasis in stage I MMMT. This process might involve integrin-α5 and does not seem to require overexpression of EGFR. Further study is required.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities.

OBJECTIVE: We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. METHODS: HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. RESULTS: HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. CONCLUSION: HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.

Effect of living cellular sheets on the angiogenic potential of human microvascular endothelial cells.

BACKGROUND: A fundamental issue limiting the efficacy of surgical approaches designed to correct periodontal mucogingival defects is that new tissues rely on limited sources of blood supply from the adjacent recipient bed. Accordingly, therapies based on tissue engineering that leverage local self-healing potential may represent promising alternatives for the treatment of mucogingival defects by inducing local vascularization. The aim of this study is to evaluate the effect of commercially available living cellular sheets (LCS) on the angiogenic potential of neonatal dermal human microvascular endothelial cells (HMVEC-dNeo). METHODS: The effect of LCS on HMVEC-dNeo proliferation, migration, capillary tube formation, gene expression, and production of angiogenic factors was evaluated over time. RESULTS: LCS positively influenced HMVEC-dNeo proliferation and migration. Moreover, HMVEC-dNeo incubated with LCS showed transcriptional profiles different from those of untreated cells. Whereas increased expression of angiogenic genes predominated early on in response to LCS, late-phase responses were characterized by up- and downregulation of angiostatic and angiogenic genes. However, this trend was not confirmed at the protein level, as LCS induced increased production of most of the angiogenic factors tested (i.e., epidermal growth factor [EGF], heparin-binding EGF-like growth factor, interleukin 6, angiopoietin, platelet-derived growth factor-BB, placental growth factor, and vascular endothelial growth factor) throughout the investigational period. Finally, although LCS induced HMVEC-dNeo proliferation, migration, and expression of angiogenic factors, additional factors and environmental pressures are likely to be required to promote the development of complex, mesh-like vascular structures. CONCLUSION: LCS favor initial mechanisms that govern angiogenesis but failed to enhance or accelerate HMVEC-dNeo morphologic transition to complex vascular structures.

Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model.

Acinar cell regeneration from tubular structures has been reported to occur in duct-deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT-PCR methods. In the duct-ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct-ligated and duct-deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT-PCR showed an increase in the epiregulin and heparin-binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.