CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett's-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions.

BACKGROUND & AIMS: Barrett's esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% of patients with Barrett's esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development. METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression targeting SCJ LGR5+ cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRASG12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). CONCLUSIONS: p16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

Changes in attachment and metabolic activity of rat neonatal cardiomyocytes and nonmyocytes caused by Macrovipera lebetina obtusa venom.

The Caucasian viper Macrovipera lebetina obtusa (MLO) is one of the most prevalent and venomous snakes in the Caucasus and the surrounding regions, yet the effects of MLO venom on cardiac function remain largely unknown. We examined the influence of MLO venom (crude and with inhibited metalloproteinases and phospholipase A2) on attachment and metabolic activity of rat neonatal cardiomyocytes (CM) and nonmyocytes (nCM), assessed at 1 and 24 h. After exposing both CM and nCM to varying concentrations of MLO venom, we observed immediate cytotoxic effects at a concentration of 100 µg/ml, causing detachment from the culture substrate. At lower MLO venom concentrations both cell types detached in a dose-dependent manner. Inhibition of MLO venom metalloproteinases significantly improved CM and nCM attachment after 1-hour exposure. At 24-hour exposure to metalloproteinases inhibited venom statistically significant enhancement was observed only in nCM attachment. However, metabolic activity of CM and nCM did not decrease upon exposure to the lower dose of the venom. Moreover, we demonstrated that metalloproteinases and phospholipases A2 are not the components of the MLO venom that change metabolic activity of both CM and nCM. These results provide a valuable platform to study the impact of MLO venom on prey cardiac function. They also call for further exploration of individual venom components for pharmaceutical purposes.

The essential role of arterial pulse in venous return.

The close proximity of arteries and veins is a well-known anatomical finding documented in the extremities of all vertebrates. However, the physiological consequences of this arrangement are rarely given proper consideration nor are they covered in the textbook list of mechanisms that aid blood flow. We hypothesized that arterial pressure pulsations can significantly increase blood flow in the adjacent valve-containing vein segments. To demonstrate the existence of this mechanism, 10- to 15-cm sections of the bovine forelimb neurovascular bundle were isolated. The proximal and distal ends of the median artery and adjacent veins were cannulated, their tributaries were tied off, and the dissected bundle was then inserted into an airtight enclosure to mimic in vivo encasement by surrounding muscle. Pulsatile pressure was subsequently applied to the arterial segment while recording venous flow. At pressure settings mimicking physiological scenarios, arterial pulsations caused a highly significant increase in venous return. The amplitude of this effect was dependent on the arterial pulsation rate, stroke volume, and pressure gradient across the vein segment.