Circulating cell-free HPV DNA is a strong marker for disease severity in cervical cancer.

For cervical cancer (CC), circulating cell-free HPV DNA (ccfHPV) may establish disease severity. Furthermore, HPV integration has been correlated to viral load and survival. In this study, pre-treatment plasma from 139 CC cases (50 primary surgery patients, 22 primary surgery + adjuvant oncological therapy patients, and 67 primary oncological therapy patients) was collected (2018-2020). Furthermore, plasma from 25 cervical intraepithelial neoplasia grade 3 patients and 15 healthy women (negative controls) were collected. Two next-generation sequencing (NGS) panels were used to establish ccfHPV presence and human papillomavirus type 16 (HPV16) integration status. ccfHPV was detected in four primary surgery (8.0%), eight primary surgery + adjuvant oncology (36.4%), and 54 primary oncology (80.6%) patients. For primary oncology patients with HPV16-related cancer (n = 37), more ccfHPVneg than ccfHPVpos patients had HPV16 integration (P = 0.04), and in patients with HPV16 integration (n = 13), ccfHPVpos patients had higher disease stages than ccfHPVneg patients (P = 0.05). In summary, ccfHPV presence is related to disease severity and may add to the debated Sedlis criteria used for identifying patients for adjuvant oncological therapy. However, ccfHPV detection is influenced by HPV integration status and disease stage, and these factors need to be considered in ccfHPVneg patients.

The Diagnostic Value of Circulating Cell-Free HPV DNA in Plasma from Cervical Cancer Patients.

Circulating cell-free HPV DNA (ccfHPV DNA) may serve as a marker for cervical cancer. In this study, we used digital droplet PCR (ddPCR) to detect and quantify ccfHPV DNA in plasma from patients with HPV16- or HPV18-associated cervical cancer. Blood samples from 60 patients diagnosed with cervical cancer (FIGO IA1-IVA) at Aarhus or Odense University Hospital (June 2018 to March 2020) were collected prior to treatment, and patients were subdivided into an early stage (n = 30) and a late-stage subgroup (n = 30) according to disease stage. Furthermore, blood samples from eight women with HPV16- or 18-associated premalignant conditions (CIN3), and 15 healthy controls were collected. ddPCR was used to analyze plasma from all participants. ccfHPV DNA was detected in 19 late-stage patients (63.33%), 3 early stage patients (10.00%), and none of the CIN3 patients or controls. Quantitative evaluation showed significant correlations between ccfHPV DNA level and stage, tumor score, and tumor size. Thus, our results indicate that ccfHPV DNA may not be a useful marker for early detection of cervical cancer. However, for patients with advanced stage cervical cancer, ccfHPV DNA level represents a promising tool to establish tumor burden, making it useful for establishing treatment response and monitoring the disease.

Anti-fibrotic mechanisms of angiotensin AT2 -receptor stimulation.

The angiotensin AT2 -receptor is a main receptor of the protective arm of the renin-angiotensin system. Understanding of this unconventional G-protein coupled receptor has significantly advanced during the past decade, largely because of the availability of a selective non-peptide AT2 -receptor agonist, which allowed the conduct of a multitude of studies in animal disease models. This article reviews such preclinical studies that in their entirety provide strong evidence for an anti-fibrotic effect mediated by activation of the AT2 -receptor. Prevention of the development of fibrosis by AT2 -receptor stimulation has been demonstrated in lungs, heart, blood vessels, kidney, pancreas and skin. In lungs, AT2 -receptor stimulation was even able to reverse existing fibrosis. The article further discusses intracellular signalling mechanisms mediating the AT2 -receptor-coupled anti-fibrotic effect, including activation of phosphatases and subsequent interference with pro-fibrotic signalling pathways, induction of matrix-metalloproteinases and hetero-dimerization with the AT1 -receptor, the TGF-ßRII-receptor or the RXFP1-receptor for relaxin. Knowledge of the anti-fibrotic effects of the AT2 -receptor is of particular relevance because drugs targeting this receptor have entered clinical development for indications involving fibrotic diseases.

Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase.

The Angiotensin II type 2 receptor (AT2R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT2R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT2R-mediated phosphorylation of eNOS at Ser1177, activation of phosphatases and dephosphorylation of eNOS at Tyr657 and Thr495 are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT2R-agonist Compound 21 (C21) (1 µM) in the presence or absence of either PD123319 (10 µM; AT2R antagonist), l-NG-Nitroarginine methyl ester (l-NAME) (10 µM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na3VO4) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser1177) or inhibitory eNOS residues (eNOS-Tyr657, eNOS-Thr495) was determined by Western blotting. Phosphorylation of Akt at Ser473 was measured to estimate Akt activity. AT2R stimulation significantly increased NO release from HAEC, which was blocked by PD123319, l-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT2R stimulation resulted in phosphorylation of eNOS-Ser1177 and dephosphorylation of eNOS-Tyr657 and eNOS-Thr495 Phosphorylation at eNOS-Ser1177 was prevented by inhibition of Akt with MK-2206. From these data, we conclude that AT2R stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr657, eNOS-Thr495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.