Correction to: Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

Correction to: Strahlenther Onkol 2018 https://doi.org/10.1007/s00066-018-1382-3 The original version of this article unfortunately contained a mistake. The correct version of the Acknowledgements is given ….

Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

BACKGROUND: Microvascular free flap reconstruction has become a standard technique in head and neck reconstructive surgery. Pre-operative radiotherapy is associated with a higher incidence of free flap malperfusion and the need for operative revision. Irradiated vessels present characteristic histomorphological and structural changes. Alterations in endothelial cells of irradiated arteries remain incompletely investigated especially with regard to long-term changes in endothelial dysfunction supporting an intraluminal pro-thrombotic and pro-inflammatory milieu. METHODS: Endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E­ and P­selectin, endothelial NO-synthase (eNOS), thrombomodulin and plasminogen activator inhibitor-1 (PAI-1) in irradiated and non-irradiated arteries was analysed using immunohistochemistry and Remmele scale grading. The average radiation dose was 58.7 ± 7.0 Gy; the time interval between end of radiation and tissue sampling was 106.0 ± 86.8 months. RESULTS: Endothelial expression of ICAM-1, VCAM-1, E­ and P­selectin as well as PAI-1 was significantly increased in previously irradiated arteries compared with non-irradiated controls, whereas thrombomodulin and eNOS expression did not show any differences. However, when comparing non-irradiated free flap arteries with irradiated arteries from the head and neck area in respective individuals, eNOS expression was significantly lower in irradiated vessels whereas ICAM-1, VCAM-1, E­/p-Selectin and PAI-1 showed significantly higher expression levels. CONCLUSION: There is ongoing endothelial dysfunction in terms of increased expression of pro-thrombotic and pro-inflammatory markers in irradiated arteries even years after radiotherapy. Treating this endothelial dysfunction might reduce the complication rates associated with microvascular free flap reconstructions in irradiated patients.

Re-expression of pro-fibrotic, embryonic preserved mediators in irradiated arterial vessels of the head and neck region.

PURPOSE: Surgical treatment of head and neck malignancies frequently includes microvascular free tissue transfer. Preoperative radiotherapy increases postoperative fibrosis-related complications up to transplant loss. Fibrogenesis is associated with re-expression of embryonic preserved tissue developmental mediators: osteopontin (OPN), regulated by sex-determining region Y­box 9 (Sox9), and homeobox A9 (HoxA9) play important roles in pathologic tissue remodeling and are upregulated in atherosclerotic vascular lesions; dickkopf-1 (DKK1) inhibits pro-fibrotic and atherogenic Wnt signaling. We evaluated the influence of irradiation on expression of these mediators in arteries of the head and neck region. MATERIALS AND METHODS: DKK1, HoxA9, OPN, and Sox9 expression was examined immunohistochemically in 24 irradiated and 24 nonirradiated arteries of the lower head and neck region. The ratio of positive cells to total cell number (labeling index) in the investigated vessel walls was assessed semiquantitatively. RESULTS: DKK1 expression was significantly decreased, whereas HoxA9, OPN, and Sox9 expression were significantly increased in irradiated compared to nonirradiated arterial vessels. CONCLUSION: Preoperative radiotherapy induces re-expression of embryonic preserved mediators in arterial vessels and may thus contribute to enhanced activation of pro-fibrotic downstream signaling leading to media hypertrophy and intima degeneration comparable to fibrotic development steps in atherosclerosis. These histopathological changes may be promoted by HoxA9-, OPN-, and Sox9-related inflammation and vascular remodeling, supported by downregulation of anti-fibrotic DKK1. Future pharmaceutical strategies targeting these vessel alterations, e. g., bisphosphonates, might reduce postoperative complications in free tissue transfer.

Osteoclast profile of medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy: a comparison with osteoradionecrosis and osteomyelitis.

BACKGROUND: The medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy [MRONJ (BP)] is characterized by non-healing exposed bone in the maxillofacial region. The pathogenesis of MRONJ (BP) is not fully understood. Giant, hypernucleated, inactive osteoclasts were found in MRONJ (BP) tissues, which indicated that accelerated cell-cell fusion might play a role. Dendritic cell-specific transmembrane protein (DC-STAMP) is associated with the cell-cell fusion of osteoclasts and precursor cells. Tartrate-resistant acid phosphatase (TRAP) is essential for osteoclastic bone resorption. The cell-cell fusion, as part of the osteoclastogenesis, and the resorptive activity can determine the morphology of osteoclasts. This study analyzed jaw bone from patients with MRONJ (BP), osteomyelitis (OM) and osteoradionecrosis (ORN) because a comparison with the osteoclast profiles of OM and ORN is essential for characterizing the osteoclast profile of MRONJ (BP). METHODS: Formalin-fixed routine jaw bone specimens from 70 patients [MRONJ (BP) n = 30; OM: n = 15, ORN: n = 15, control: n = 10] were analyzed retrospectively for osteoclast quantity, morphology and the expression of TRAP and DC-STAMP. The specimens were processed for hematoxylin and eosin staining (H&E), histochemistry (TRAP) and immunohistochemistry (anti-DC-STAMP) and were analyzed via virtual microscopy. RESULTS: The quantity, diameter and nuclearity of osteoclasts were significantly higher in MRONJ (BP) specimens than in OM, ORN and control specimens. Giant, hypernucleated osteoclasts were detected in MRONJ (BP) specimens only. Osteoclastic TRAP expression was lower in MRONJ (BP) and ORN specimens than in OM and control specimens. The DC-STAMP expression of osteoclasts and mononuclear cells was significantly higher in MRONJ (BP) and ORN specimens than in OM and control specimens. CONCLUSIONS: This study indicates that the osteoclast profile of MRONJ (BP) is characterized by osteoclast inactivation and a high cell-cell fusion rate; however, the presence of giant, hypernucleated osteoclasts cannot be attributed to increased DC-STAMP-triggered cell-cell fusion alone. The incidental characterization of the osteoclast profiles of OM and ORN revealed differences that might facilitate the histopathological differentiation of these diseases from MRONJ (BP), which is essential because their therapies are somewhat different.

BRONJ-related jaw bone is associated with increased Dlx-5 and suppressed osteopontin-implication in the site-specific alteration of angiogenesis and bone turnover by bisphosphonates.

OBJECTIVES: Site-specific suppression of bone remodelling has been implicated in bisphosphonate-(BP)-related osteonecrosis of the jaws (BRONJ). Due to the origin of jaw bone from cranial neural crest, osseous differentiation is regulated specifically by the antagonizing BMP-2-downstream-transcription factors Msx-1 and Dlx-5. Osteopontin has been implicated in bone remodelling and angiogenesis. The osteoblast and osteoclast progenitor proliferation mediating Msx-1 has been demonstrated to be suppressed in BRONJ. In vitro BPs were shown to increase Dlx-5 and to suppress osteopontin expression. This study targeted Dlx-5 and osteopontin in BRONJ-related and BP-exposed jaw bone compared with healthy jaw bone samples at protein- and messenger RNA (mRNA) level, since increased Dlx-5 and suppressed osteopontin might account for impaired bone turnover in BRONJ. MATERIALS AND METHODS: Fifteen BRONJ-exposed, 15 BP-exposed and 20 healthy jaw bone samples were processed for real-time reverse transcription polymerase chain reaction (RT-PCR) and for immunohistochemistry. Targeting Dlx-5, osteopontin and glyceraldehyde 3-phosphate dehydrogenase mRNA was extracted, quantified by the LabChip-method, followed by quantitative RT-PCR. For immunohistochemistry, an autostaining-based alkaline phosphatase antialkaline phosphatase (APAPP) staining kit was used. Semiquantitative assessment was performed measuring the ratio of stained cells/total number of cells (labelling index, Bonferroni adjustment). RESULTS: The labelling index was significant decreased for osteopontin (p < 0.017) and significantly increased for Dlx-5 (p < 0.021) in BRONJ samples. In BRONJ specimens, a significant fivefold decrease in gene expression for osteopontin (p < 0.015) and a significant eightfold increase in Dlx-5 expression (p < 0.012) were found. CONCLUSIONS: BRONJ-related suppression of bone turnover is consistent with increased Dlx-5 expression and with suppression of osteopontin. The BP-related impaired BMP-2-Msx-1-Dlx-5 axis might explain the jaw bone specific alteration by BP. CLINICAL RELEVANCE: The findings of this study help to explain the restriction of RONJ to craniofacial bones. BRONJ might serve as a model of disease elucidating the specific signal transduction of neural crest cell-derived bone structures in health and disease.

Expression of transforming growth factor beta 1-related signaling proteins in irradiated vessels.

AIM: Microvascular free tissue transfer is a standard method in head and neck reconstructive surgery. However, previous radiotherapy of the operative region is associated with an increased incidence in postoperative flap-related complications and complete flap loss. As transforming growth factor beta (TGF-ß) 1 and galectin-3 are well known markers in the context of fibrosis and lectin-like oxidized low-density lipoprotein 1 (LOX-1) supports vascular atherosclerosis, the aim of this study was to evaluate the expression of TGF-ß1 and related markers as well as LOX-1 in irradiated vessels. MATERIALS AND METHODS: To evaluate the expression of galectin-3, Smad 2/3, TGF-ß1, and LOX-1, 20 irradiated and 20 nonirradiated arterial vessels were used for immunohistochemical staining. We semiquantitatively assessed the ratio of stained cells/total number of cells (labeling index). RESULTS: Expression of galectin-3, Smad 2/3, and TGF-ß1 was significantly increased in previously irradiated vessels compared with nonirradiated controls. Furthermore, LOX-1 was expressed significantly higher in irradiated compared with nonirradiated vessels. CONCLUSION: Fibrosis-related proteins like galectin-3, Smad 2/3, and TGF-ß1 are upregulated after radiotherapy and support histopathological changes leading to vasculopathy of the irradiated vessels. Furthermore, postoperative complications in irradiated patients can be explained by increased endothelial dysfunction caused by LOX-1 in previously irradiated patients. Consequently, not only TGF-ß1 but also galectin-3 inhibitors may decrease complications after microsurgical tissue transfer.