Earth--moon evolution: implications for the mechanism of the biological clock?

The geophysical characteristics of the planet Earth dictate the physiological traits of living organisms. Changes in the geophysical conditions over the course of geological time are responsible for major evolutionary changes in life emergence and evolvement. Calendar day length is one of earth's geophysical characteristics which is under a constant, if extremely small, progressive change. This enforces an adjustment of circadian rhythmicity throughout geological time. The calendar day has extended approximately 9 hours in the last 3.5 billion years. Two mechanisms for circadian-rhythm adjustment are suggested: a directional selection mechanism -- an endogenous -- oriented explanation regarding a genetic drift in the population's endogenous oscillation toward a lengthened daily cycle; and an exogenous calibration mechanism - a hypothesis on the existence of a geophysical responsive element which senses a geophysical stimuli and calibrates the inner cellular oscillation in accordance with the length of the calendar day. A distinguishing experiment between the two explanations is suggested and discussed. Circadian rhythm mechanism and the evolution of circadian rhythmicity are tightly connected. Circadian rhythms' evolutionary theories are discussed in light of their contribution to our understanding of the selective pressures being applied throughout geological time and of how, once the clock has been established, it maintains an ongoing adjustment to a continuous change in the length of day.I argue that the exogenous calibration mechanism combines with the endosymbiont coordination theory, together, present an explanation to the path by which the calendar day adjustment was acquired and maintained. This hypothesis suggests a role for gravity cyclic force and for cytoskeleton's components in calendar day adjustment mechanism and circadian rhythm entrainment.

The physical imperative in circadian rhythm: a cytoskeleton-related physically resettable clock mechanism hypothesis.

Organisms maintaining circadian rhythmicity are responding to physical constraint of a 24-hour cycle. Time-cue sensing is fundamental to the clock existence, and entrainment of circadian rhythm is indeed accessible to a wide variety of geophysical stimuli. Light-dark and temperature changes are the main time-cues. Additional physical forces such as barometric pressure, electrostatic and electromagnetic fields and gravity force, display a daily cyclic behavior and can function as secondary time-cues. A conceptual framework that contains explanations to all circadian properties including cell autonomous, environmental responsiveness and self-sustained character, is still lacking. It is argued that clock responsiveness to external cues is central to the cellular clock mechanism, and therefore, the nature of the time-cues and the pathways that enable the cell to respond to physical stimuli are of central importance. A role for cytoskeleton in clock entrainment mechanism is suggested in light of cytoskeleton's major involvement in cellular mechanotransduction.

Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases.

BACKGROUND: It is generally assumed that unwarranted, excessive neovascularization of the retina and iris is a direct response to a hypoxic retinal environment. Prompted by our previous findings that the potent angiogenic factor, vascular endothelial growth factor (VEGF), is hypoxia-inducible, we used in situ hybridization techniques to examine the thesis that VEGF functions as the link between retinal ischemia and a pathologic, intraocular, angiogenic response. EXPERIMENTAL DESIGN: To gain molecular access to human material representing progressive stages of angiogenic eye diseases, in situ hybridization analysis was carried out on sections of whole globes enucleated at the time of ongoing neovascularization. This methodology identified cells that have up-regulated VEGF expression during natural progression of the indicated diseases. A rabbit model was also used to determine whether experimentally induced retinal ischemia leads to up-regulation of VEGF expression. RESULTS: Proliferation of vascular elements in proliferative diabetic retinopathy and neovascularization of the retina and/or iris secondary to central retinal vein occlusion, retinal detachment, and intraocular tumors were always accompanied by induction of retinal VEGF expression. Furthermore, in each case, expression of VEGF was induced only in a particular layer of the retina (either the outer nuclear layer, the inner nuclear layer, or the ganglion cell layer), matching the zones affected by impaired perfusion. In a rabbit model simulating retinal vein occlusion, elevated levels of VEGF mRNA were detected within a few days of experimental induction of retinal ischemia, exclusively in the ischemic region. CONCLUSIONS: VEGF may be one of the long anticipated factors linking retinal ischemia and intraocular angiogenesis. Irrespective of the cause of retinal ischemia, sustained overproduction of VEGF by ischemic retinal cells may promote retinal and iris neovascularization in a number of neovascular eye diseases.

Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis.

Perfusion insufficiency, and the resultant hypoxia, often induces a compensatory neovascularization to satisfy the needs of the tissue. We have used multicellular tumor spheroids, simulating avascular microenvironments within a clonal population of glioma tumor cells, in conjunction with in situ analysis of gene expression, to study stress inducibility of candidate angiogenic factors. We show that expression of vascular endothelial growth factor (VEGF) is upregulated in chronically hypoxic niches (inner layers) of the spheroid and that expression is reversed when hypoxia is relieved by hyperoxygenation. Acute glucose deprivation--another consequence of vascular insufficiency--also activates VEGF expression. Notably, glioma cells in two distinct regions of the spheroid upregulated VEGF expression in response to hypoxia and to glucose starvation. Experiments carried out in cell monolayers established that VEGF is independently induced by these two deficiencies. Upon implantation in nude mice, spheroids were efficiently neovascularized. Concomitant with invasion of blood vessels and restoration of normoxia to the spheroid core, VEGF expression was gradually downregulated to a constitutive low level of expression, representing the output of nonstressed glioma cells. These findings show that stress-induced VEGF activity may compound angiogenic activities generated through the tumor "angiogenic switch" and suggest that stress-induced VEGF should be taken into account in any attempt to target tumor angiogenesis.

Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis.

OBJECTIVE: The process of coronary collateral development is poorly understood. It is assumed that particular angiogenic factors are upregulated during episodes of myocardial ischaemia and act as a trigger for neovascularisation. However, the identity of these factors is unknown. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be hypoxia inducible, so this factor may mediate ischaemia induced angiogenesis in the heart. The aim of this study was to examine hypoxia inducibility of VEGF in cultured myocardial cells as well as in normally perfused and ischaemic porcine myocardium. METHODS: (1) In vitro experiment: cultured rat myocardial cells were subjected to hypoxia, and steady state levels of VEGF mRNA were measured after 2 and 4 h of hypoxia. (2) In vivo experiment: myocardial ischaemia in pigs hearts was induced by repeated 2-10 min left anterior descending coronary artery occlusions, separated by 20 min of reperfusion. Hearts were retrieved after 6 h of intermittent ischaemia. Total RNA was extracted from normal and ischaemic zones of the heart and processed for RNA blot hybridisation analysis. RESULTS: In vitro experiment: as soon as 2-4 h after exposure of cultures to hypoxia, VEGF mRNA levels were significantly raised (6-10-fold). In vivo experiment: VEGF expression was significantly augmented in the ischaemic territory of the myocardium (three- to fivefold induction). Furthermore, polymerase chain reaction amplification of the reverse transcribed mRNA showed increased production of multiple forms of differentially spliced VEGF mRNA in the ischaemic myocardium. CONCLUSIONS: VEGF production in the myocardium is significantly upregulated by hypoxia in vitro and by ischaemia in vivo. These results suggest that VEGF is a likely mediator in the natural process of ischaemia induced myocardial neovascularisation.

Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis.

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific mitogen. To evaluate whether VEGF may play a role in angiogenesis, we have determined the spatial and temporal patterns of expression of VEGF and VEGF receptors during natural angiogenic processes taking place within the female reproductive system. Four angiogenic processes were analyzed: neovascularization of ovarian follicles, neovascularization of the corpus luteum, repair of endometrial vessels, and angiogenesis in embryonic implantation sites. During all processes, VEGF mRNA was found to be expressed in cells surrounding the expanding vasculature. VEGF was predominantly produced in tissues that acquire new capillary networks (theca layers, lutein cells, endometrial stroma, and the maternal decidua, respectively). VEGF-binding activity, on the other hand, was found on endothelial cells of both quiescent and proliferating blood vessels. These findings are consistent with a role for VEGF in the targeting of angiogenic responses to specific areas. Using in situ hybridization, we show that VEGF is expressed in 10 different steroidogenic and/or steroid-responsive cell types (theca, cumulus, granulosa, lutein, oviductal epithelium, endometrial stroma, decidua, giant trophoblast cells, adrenal cortex, and Leydig cells). Furthermore, in some cells upregulation of VEGF expression is concurrent with the acquisition of steroidogenic activity, and expression in other cell types is restricted to a particular stage of the ovarian cycle. These findings suggest that expression of VEGF is hormonally regulated. We propose that excessive expression of VEGF during gonadotropin-induced ovulation may contribute to the development of ovarian hyperstimulation syndromes by virtue of the vascular permeabilization activity of this factor.

Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.

Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. Examples include the compensatory development of collateral blood vessels in ischaemic tissues that are otherwise quiescent for angiogenesis and angiogenesis associated with the healing of hypoxic wounds. But the presumptive hypoxia-induced angiogenic factors that mediate this feedback response have not been identified. Here we show that vascular endothelial growth factor (VEGF; also known as vascular permeability factor) probably functions as a hypoxia-inducible angiogenic factor. VEGF messenger RNA levels are dramatically increased within a few hours of exposing different cell cultures to hypoxia and return to background when normal oxygen supply is resumed. In situ analysis of tumour specimens undergoing neovascularization show that the production of VEGF is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions) and the clustering of capillaries alongside VEGF-producing cells.