Apoptosis a programmed cell death, is an essential mechanism of eliminating damaged or aged cells and thus to maintain tissue integrity. There are two central pathways that lead to apoptosis: a) the positive induction by ligands (death factors) binding to plasma membrane receptors (death factor receptors) and b) negative induction by the loss of suppressor activity. The common execution mechanisms of apoptosis consist of the activation of cytosolic aspartate-specific proteases (ICE-proteases) termed caspases, which can be activated via various intracellular pathways. In the stomach, mucosal surface epithelial cells are constantly exfoliating to the gastric lumen and completely replaced within 3-5 days under physiological conditions. Apoptosis has been reported to take place in all regions of the stomach with apoptotic cells occurring predominantly in the superficial parts of the gastric glands, at a rate of 2-3% for all cells. Following mucosal injury (e.g. ulcer development), apoptosis rapidly increases and remains elevated for 2-3 months. In a 3-month old ulcer scar, the apoptosis rate of mucous, parietal, chief and endocrine cells was found to be similar to that of normal gastric mucosa. Helicobacter pylori (H. pylori) infection induces apoptosis in the gastric mucosa and this action appears to be independent of VacA cytotoxin of H. pylori strains. Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) inhibitors are potent inductors of gastric epithelial cell apoptosis. However, they can abrogate apoptosis or proliferation effects induced by H. pylori. Many details of the exact intracellular and molecular mechanisms regulating apoptosis in gastric mucosa remain to be elucidated.

Apoptosis a programmed cell death, is an essential mechanism of eliminating damaged or aged cells and thus to maintain tissue integrity. There are two central pathways that lead to apoptosis: a) the positive induction by ligands (death factors) binding to plasma membrane receptors (death factor receptors) and b) negative induction by the loss of suppressor activity. The common execution mechanisms of apoptosis consist of the activation of cytosolic aspartate-specific proteases (ICE-proteases) termed caspases, which can be activated via various intracellular pathways. In the stomach, mucosal surface epithelial cells are constantly exfoliating to the gastric lumen and completely replaced within 3-5 days under physiological conditions. Apoptosis has been reported to take place in all regions of the stomach with apoptotic cells occurring predominantly in the superficial parts of the gastric glands, at a rate of 2-3% for all cells. Following mucosal injury (e.g. ulcer development), apoptosis rapidly increases and remains elevated for 2-3 months. In a 3-month old ulcer scar, the apoptosis rate of mucous, parietal, chief and endocrine cells was found to be similar to that of normal gastric mucosa. Helicobacter pylori (H. pylori) infection induces apoptosis in the gastric mucosa and this action appears to be independent of VacA cytotoxin of H. pylori strains. Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) inhibitors are potent inductors of gastric epithelial cell apoptosis. However, they can abrogate apoptosis or proliferation effects induced by H. pylori. Many details of the exact intracellular and molecular mechanisms regulating apoptosis in gastric mucosa remain to be elucidated.

Gastric mucosal microvessels were isolated after arterial perfusion of the rat stomach with magnetized iron oxide suspension. After homogenization of scrapped gastric mucosa, microvessels were initially separated with a high power magnet and further separated and purified by using a nylon sieve. Aliquots of purified microvessels were assessed for viability, histologic appearance, ultrastructure and generation of prostacyclin. Microvessels were plated on Matrigel and cultured in DMEM with high glucose and 10% FBS for 1, 3 or 5 days. After 1, 3 and 5 days of culturing, endothelial viability was assessed with Fast green exclusion, and the basal and stimulated (with calcium ionophore) generation of prostacyclin was determined by assaying aliquots of the incubating medium for 6-keto PGF(1alpha). At 1 and 3 hrs after isolation, microvessels demonstrated intact morphologic structures as reflected by transmission EM and 92+/-4% of viable endothelial cells. The microvessels plated on Matrigel maintained good viability for at least 5 days and generated prostacyclin at the baseline and following ionophore stimulation. These data demonstrate that isolated microvessels cultured under optimal conditions are fully viable and functional.

The effects of exogenous ATP on Ca2+ signaling and wound healing were investigated in rat gastric microvascular endothelial cells (RGMEC). ATP (10 microM) triggered a significant rise in intracellular Ca2+ concentration ([Ca2+]i) from 46+/-2 nM at baseline to peak values averaging 283+/-31 nM (n = 5 experiments, 132 cells). Return to the basal [Ca2+]i was delayed by slowly declining plateau phase that persisted for 200+/-30 s. Removal of extracellular Ca2+ did not significantly affect the peak rise in [Ca2+]i, but reduced the plateau. ATP (10 microM) also significantly increased the migration of RGMEC in a wounded monolayer. Addition of the non-subtype selective purinergic receptor antagonist, suramin, abrogated the effects of ATP on [Ca2+]i and migration. We conclude that local elevation of ATP acting through purinergic receptors induce Ca2+ signals in RGMEC and may contribute to endothelial cell migration.

We have previously characterized morphologic features of wounding-induced angiogenesis that occurs in response to acute and chronic gastric mucosal injury. As a means of investigating the molecular mechanisms underlying gastric angiogenesis, microvascular endothelial cells were isolated from stomachs of normal (non-injured) rats. The isolation procedure adapted and combined aspects of previous methods and employed positive selection using magnetic beads coated with monoclonal antibody specific for rat CD31 (PECAM-1), a cell surface marker restricted to platelets, monocytes, T lymphocytes and endothelial cells. The isolated microvascular endothelial cells expressed vascular endothelium-specific antigen and the endothelial-specific receptors, Tie2 and flt-1 (VEGFR1). When plated on growth factor-reduced matrigel, the isolated microvascular endothelial cells formed capillary-like structures reflecting in vitro angiogenesis. These cells were also responsive to vascular endothelial growth factor, VEGF, further verifying their endothelial nature. The rat microvascular endothelial cells isolated by this procedure should be useful in delineating molecular mechanisms and regulation of the angiogenesis that is essential for the healing of acute and chronic gastric injury.