My current research is primarily in the area of

Orthostasis

Orthostasis (upright posture) is a fundamental human activity requiring rapid and effective circulatory and neurologic compensations in order to maintain blood pressure and consciousness. If not for these defense mechanisms the precarious positioning of the brain well above the neutral cardiac point (roughly at the right atrium) and the presence of large venous reservoirs below the neutral point would cause blood pressure to fall rapidly due to gravitational pooling of blood within the dependent veins with decreased brain blood flow and loss of consciousness.

Our primary defense against pooling is the “muscle pump” in which contractions of leg muscles propels sequestered venous blood back to the heart. This also encourages forward flow by reducing venous pressure and increasing the pressure difference between leg arteries and leg veins. The compression of vessels during skeletal muscle contraction expels both venous blood and lymphatic fluid from the lower extremities. Immediately after contraction, both veins and lymphatic vessels are relatively empty, which can be shown by direct measurement of hyaluronan as marker for lymphatic outflow. A defective muscle pump is an important reason that astronauts are vulnerable to orthostatic stress after exposure to low gravity; they develop rapid leg muscle atrophy and refractory lower limb pooling. The muscle pump is also partly defeated during quiet standing and is nearly completely defeated while standing without motion.

The second line of defense against orthostatic intolerance is neurovascular adjustment which includes rapid changes in artery resistance vessel tone (vasoconstriction) limiting flow to the extremities and splanchnic (liver, spleen and digestive tract) vascular bed while promoting passive emptying. The splanchnic bed is the largest venous reservoir at least when supine. Splanchnic venoconstriction occurs further enhancing emptying. These reflex compensatory mechanisms are primarily controlled by the high-pressure arterial pressure receptors also called “arterial baroreceptors” and to a lesser degree by low-pressure cardiopulmonary reflexes.

Additionally, humoral effects may enhance the defense against standing through the quick release of epinephrine, vasopressin, the recently discovered peptide adrenomedullin, and by central effects. Later, activation of the renin-angiotensin-aldosterone system may enhance the long-term defense against standing by fluid retention within the kidney. The ultimate benefit of this is that an increase in blood volume generally reduces the magnitude of the reflex alterations which are required to tolerate upright posture, as during quiet standing the neurovascular compensatory mechanisms are only partly effective.

Adrenomedullin (ADM)

Adrenomedullin is one of the most potent hypotensive agents known, exerting most of its vasodilatory effects via production of cAMP and consequently generation of nitric oxide. This 52 amino acid peptide is produced in multiple tissues. Most notable both the endothelial and smooth muscle components of the vasculature, the adrenal medulla and the central nervous system and many of its actions seem related to the homeostatic control of fluid and electrolyte status.

ADM is a potent natriuretic agent particularly by inhibition of aldosterone release in response to angiotensin II and potassium. In animals the hypotensive effect of intravenous ADM is accompanied by increased renin secretion, but no significant change in vasopressin release. In addition, to his hypotensive capability this peptide exerts positive inotropic and chronotropic effects conceivably as protective factor against vascular collapse during shock. Similarly, some publications indicate that ADM may buffer the sympathetic response to hypertension. On the other hand, direct CNS actions of ADM by stimulating sympathetic activity are proposed.

Besides inhibition of ACTH and CRH release – interestingly without generation of cAMP - central administration of ADM inhibits water drinking in response to physiological stimuli. Such central injections of ADM or even PAMP elevate mean arterial blood pressure and inhibit vasopressin release and salt appetite. Although the exact site of action in brain is unknown, the rapidity of the effect suggests a hypothalamic action perhaps by stimulating sympathetic nervous system. Analogous to the positive inotropic effects of ADM in the heart, centrally produced ADM as well as PAMP may be part or a protective system maintaining vascular competence and adequate perfusion pressures to organs.

Proadrenomedullin N-Terminal 20 Peptide (PAMP)

PAMP consists of 20 amino acids and its distribution is similar to that of human ADM because both peptides are biosynthesized from an common precursor. Similarly to ADM PAMP elicits a potent hypotensive effect in a dose dependent manner after intravenous injection but in contrast to ADM this effect lasts only for a short time. While ADM exerts its vasorelaxant affect via multiple actions in the endothelial cell layer and directly on the vascular smooth muscle cells, PAMP expresses its hypotensive action via presynaptic inhibition of sympathetic neurons innervating the vessels. Thus the hypotensive effect seems to be due to an inhibition of neural transmission at nerve endings rather than via a direct vasodilating effect.

Additionally, PAMP acts within the adrenal medulla, as in the vasculature, to reduce catecholamine release in response to sympathetic activation. Comparable to ADM, PAMP inhibits significantly the release of aldosterone from human adrenal cells induced by angiotensin II and potassium, whereas the effect of ADM is less potent than of PAMP. Summarizing these actions, PAMP seems to be able in desensitizing the baroreceptor reflex as well as lowering blood pressure by relaxing vascular smooth muscles.

Physiological adaptations during weightlessness or bedrest

The common similarity shared by weightlessness and bedrest is the loss of hydrostatic fluid columns along the long axis of body. Prolonged bedrest is associated with distinct changes in body fluid volumes, volume distribution, and deterioration of normal reflex mechanisms responsible for peripheral vascular control. When moving from an upright to a supine position, approximately 700-900 ml of blood redistribute from the lower body into the central circulation. Most of the fluid is diverted to the heart and lungs, increasing pulmonary blood flow by up to 30% resulting in augmented stroke volume, decreased heart rate and peripheral vascular resistance. This acute autonomic regulatory response, the decreased sympathetic nervous function, can be seen in depressed plasma and urinary levels of catecholamines. These counterregulation ensure to maintain mean arterial blood pressure despite regional changes in vascular pressures. Most of the cardiovascular changes occurring with weightlessness have also been shown to happen with bedrest. Therefore it is evident that bedrest offers an opportunity to study the mechanisms underlying deconditioning and to develop and screen appropriate countermeasures.

Initial fluid shifts during bedrest have been observed and measured by several scientists using different techniques. Thoracic impedance decreases immediately after head-down tilting, but returns toward pretilt within 48 hours. Urine output, especially for the first 6 hours is increased. Cardiovascular changes seen with bedrest include consistent decreases of 15-20% in plasma volume and 5-10% in blood volume. Furthermore, a sodium diuresis during the first few days of bedrest can be observed. After 6 hours of bedrest total blood volume is decreased by about 500 ml and after one day total body water is reduced by 1.3 l. This mechanism occurs within the first few hours of bedrest and reaches a maximum after 4-8 hours. Within 24 hours, urinary flow returns to the pre-bedrest level. Concurrent with decreased plasma volume an increase in hematocrit indicates hemoconcentration, which may be mediated by the Henry-Gauer reflex and by secretion of ANP during the volume overload of the central circulation. Reduced levels of plasma renin activity, and decreased levels of aldosterone and vasopressin were observed, but hormone levels returned back to control values within 24 hours. In contrast elevated plasma renin activity and depressed aldosterone levels after 6 day of 6° head-down bedrest are observed, indicating a change in the coupling of these two hormones. Even increased levels in plasma renin activity (PRA) and aldosterone are described. Despite the persistently negative sodium and potassium balance and the reductions in plasma volume, PRA and aldosterone secretory rate and thirst sensation do not increase. Taken together the diverging results demonstrate that no clear answer can be found measuring hormone values during simulated weightlessness as well as space flight and conflicting results may accompanied by different analytical techniques.

Another important function in controlling renal handling of salt and water may be contributed to the atrial natriuretic peptide. ANP is a peptide hormone discovered in 1981 and believed to have an important function upon homeostasis of plasma volume. It is synthesized in muscle cells of the atria in heart, stored there in granules, and released into the bloodstream by stretching atrial walls. It is rapidly degraded by a neutral endopeptidase and removed from the bloodstream after specific binding to a widely distributed ANP clearance receptor or cANP receptor. This hormone was characterized initially in bovine aortic smooth muscle and acts on early renal tubules sites to enhance the renal excretion of both salt and water by the kidney. Furthermore, it is suggested to play a key role in lowering vascular resistance thereby acting as a regulator of transvascular fluid balance. ANP is able to modulate microvascular permeability and to regulate the partition of extracellular fluid between the interstitial compartment and the circulating plasma. These mechanisms lead in the establishment of a new steady state resulting in decreased plasma volume, reduced cardiac output and in lowering arterial pressure.

The cellular events responsible for ANP-induced increases in vascular permeability might be related to two different types of cell-surface receptors which can specifically interact with the hormone. Binding of ANP to the ANP-R1 receptor subtype exhibits intrinsic guanylate cyclase activity resulting in an elevation of intracellular 3´,5´ß-cyclic monophosphate (cGMP). This subtype accounts for about 10% of the total ANP receptor population, whereas more than 90% of the ANP receptors on endothelial cells are the low molecular weight subtype ANP-R2 (C-receptor). This receptor subtype is characterized by the lack of guanylate cyclase production upon stimulation with ANP thus no elevation of cellular cGMP can be observed. It originally was proposed that this receptor subtype contributes to clearance of ANP from plasma, although activation of ANP-R2 receptors are able to induce cellular responses, e.g. inhibition of adenylate cyclase and reduction of intracellular cAMP concentration. Therefore, activation of both receptor subtypes might induce a decrease of cellular cAMP, whereas ANP-R2 directly inhibits adenylate cyclase and ANP-R1 indirectly inhibits cAMP stimulation through cGMP-stimulated phosphodiesterase. In this context it is noteworthy that during long term spaceflight a permanent decrease in plasma cGMP concentration could be observed.

Lymph flow

The lymphatic system represents a system which enables large molecules that have inadvertently leaked into the interstitial space to reenter the circulating blood. Capillaries of this system are very porous can thereby collect easily large particles accompanied by interstitial fluid. The so called lymph is filtered by through lymph nodes where partly large molecules like hyaluronan up to 90% are degraded, and reenters the circulatory system near the right heart. Roughly 2.5 liters of lymphatic fluid enters the cardiovascular system each day, which prevents severe swelling of interstitial space by removing excess capillary filtrate from the tissues. The interstitial space may be defined as the space located between the capillary walls and the cells. The basic structure is similar in all tissues: collagen builds the fiber framework that contains a gel phase made up of glycosaminoglycans (GAGs), a salt solution, and proteins derived from plasma. The amount of interstitium varies from about 50% of wet weight in skin to 10% in skeletal muscle. GAGs and their major component hyaluronan are of importance for interstitial fluid volume control. Hyaluronan contributes to the gel-like structure of the interstitium and is in osmotic equilibrium with collagen and proteins in the interstitial fluid. Variation in the content and concentration of hyaluronan may therefore affect interstitial fluid characteristics like hydrostatic pressure and volume. In addition, changes in GAG content may also be of importance in disease conditions affecting the connective tissue.

Hyaluronan as marker for altered lymph flow

Hyaluronan (HA) is a widely distributed extracellular negatively charged unbranched polysaccharide characterized by a highly polymerized chain of glucuronic acid and N-acetylglucosamine units (see figure below) and because it is one of the most hydrophilic molecules in nature HA is often described as "nature’s moisturizer".

When incorporated into a neutral aqueous solution hydrogen bond formation occurs between water molecules and adjacent carboxyl and N-acetyl groups. The hydrogen bond formation results in the unique water-binding and retention capacity of the polymer which is im. It also follows that the water-binding capacity is directly related to the molecular weight of the molecule. Up to six liters of water may be bound per gram of HA!!! The molecule serves in bone formation, cartilage structural maintenance, lubrication, wound healing, tissue transport, and is able to interact with the immune system in a molecular weight dependent fashion.

One important function of HA is its ability to immobilize certain molecules in the desired location of the body (aggrecan, versican, neurocan, brevican, CD44, RHAMM, HARE, LYVE-1), these interactions are essential to the structure and assembly of several tissues. The networks that HA forms are efficient insulators, since other macromolecules have trouble in finding room inside the HA network. With this property HA (and other polysaccharides) can regulate e.g. the distribution and transport of major part of plasma proteins into the tissues. Additionally, HA has been reported to be involved in various events during morphogenesis and differentiation. Its concentrations increase in the areas, where cell migration begins, suggesting that HA opens some paths through which the cells can migrate. Cancer cells are often enriched with HA, and intense intracellular staining for HA is a poor prognostic indicator for cancer therapy.

The production of HA is increased during inflammation, and generally, the viscous solutions seem to inhibit the cell activities. Hyaluronan seems to increase phagocytosis in monocytes and granulocytes, but the importance of this phenomenon is unknown.

Extracellular HA concentration far exceeds that in the bloodstream because of rapid degradation by hepatic and other endothelium after lymphatic discharge. Thus, the intravascular hyaluronan pool is being constantly removed, whereas the extravascular pool serves its physiological tissue functions. Plasma levels for HA ranges from 10 to 100 µg/l in adults aged 20-60, with a mean between 30-40 µg/l. The average molecular weight in human plasma is in the range of 140 kD, making it all but impermeable to most capillary walls. Part of the circulating hyaluronan is possibly catabolized in the kidney and spleen. Experiments with radioactively labeled hyaluronan have shown that it is rapidly turned over from the circulation. The endothelial cells of the liver also degrade hyaluronan. These cells have a specific receptor for the endocytosis of hyaluronan that also recognizes chondroitin sulfate with a 3-fold affinity compared to hyaluronan. After endocytosis, hyaluronan is transported into the lysosomes, where specific enzymes (hyaluronidase, b-glucuronidase and b-N-acetylglucosaminidase) degrade it into monosaccharides.

The flow of lymph is primarily determined by the outward flow of water from blood plasma to tissue fluid. But it is not clear how much, under physiological circumstances, postural changes, physical exercise, and/or gastrointestinal activity contribute to plasma HA variation in normal humans.

Interstitial hyaluronan concentration usually is 1-2 orders of magnitude higher than plasma HA, with considerable interindividual differences. Therefore, increased lymphatic propulsion can be expected to increase plasma levels of HA. Hyaluronan output from the tissue spaces via the lymphatics is assisted by muscular activity, or other stimuli like orthostasis, which also elevates interstitial pressure. For example slight body tilting or assumption of the upright position after prolonged rest is followed by a 3-5-fold rise in the mean lymph flow. This effect lasts for about one hour, and then a slight decrease can be observed. A high flow of concentrated lymph is observed during the first hour of standing position, conceivably because venous expansion pushes interstitial fluid into the lymphatic system. A delayed and gradual increase of lymph flow seems to be a response to orthostasis and local peripheral stasis.