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Nitric oxide-sensitive guanylyl cyclase (NO-GC) is a heterodimeric enzyme with an α- and a β-subunit. In its active form as an α 1 β 1 -heterodimer, NO-GC produces cyclic guanosine-3',5'-monophophate (cGMP) to regulate vasodilation and proliferation of vascular smooth muscle cells (VSMCs). In contrast to VSMCs, only a few studies reported on the expression of the NO-GC α 1 β 1 -heterodimer in human pericytes. Since NO-GC is a marker for platelet-derived growth factor-β (PDGFRβ)-positive pericytes, we investigated whether NO-GC is expressed in its active α 1 β 1 -heterodimer in pericytes of healthy human dental pulp. In our previous studies, we developed and validated an antibody against the α 1 -subunit of human NO-GC. Here, we developed a new antibody against the β 1 -subunit of human NO-GC and validated it by immunoblot, mass spectrometry, and immunohistochemistry on tissue samples from humans and NO-GC knockout (GCKO) mice. Using both antibodies, we detected α 1 - and β 1 -subunits of NO-GC in pericytes of pre-capillary arterioles, capillaries, and post-capillary venules in dental pulp of decalcified and non-decalcified human molars. We concluded that NO-GC as an active α 1 β 1 -heterodimer may be involved in the regulation of vascular permeability, vascular stability, organ homeostasis, and organ regeneration in healthy human dental pulp.

The binding of nitric oxide (NO) to heme in the β 1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α 1 β 1 and α 2 β 1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α 1 and β 1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α 1 and β 1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe 2+ to Fe 3+ in the β 1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.

VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells ( p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions ( p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.

Immune checkpoint inhibitors (ICI) represent a new therapeutic approach in recurrent and metastatic head and neck squamous cell carcinoma (HNSCC). The patient selection for the PD-1/PD-L1 inhibitor therapy is based on the degree of PD-L1 expression in immunohistochemistry reflected by manually determined PD-L1 scores. However, manual scoring shows variability between different investigators and is influenced by cognitive and visual traps and could therefore negatively influence treatment decisions. Automated PD-L1 scoring could facilitate reliable and reproducible results. Our novel approach uses three neural networks sequentially applied for fully automated PD-L1 scoring of all three established PD-L1 scores: tumor proportion score (TPS), combined positive score (CPS) and tumor-infiltrating immune cell score (ICS). Our approach was validated using WSIs of HNSCC cases and compared with manual PD-L1 scoring by human investigators. The inter-rater correlation (ICC) between human and machine was very similar to the human-human correlation. The ICC was slightly higher between human-machine compared to human-human for the CPS and ICS, but a slightly lower for the TPS. Our study provides deeper insights into automated PD-L1 scoring by neural networks and its limitations. This may serve as a basis to improve ICI patient selection in the future.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α 1 - and β 1 -subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α 1 - and β 1 -subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α 1 - and β 1 -subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.