Effects of gravity on proliferation and differentiation of adipose tissue-derived stem cells.

Recent studies have demonstrated that culturing stem cells under altered gravity conditions modulates their proliferation and differentiation. In the current study we focused on osteogenesis. In an attempt to induce high proliferation rates and low differentiation of adipose tissue-derived stem cells (AT-MSCs), we exposed them to simulated microgravity (sim-microg) and hypergravity. We used the random positioning machine (RPM) to simulate microg and the medium sized centrifuge for acceleration research (MidiCAR) for hypergravity. AT-MSCs from different origin (human and goat) seeded in OptiCells were housed in the RPM and MidiCAR and compared with suitable controls cultured under static conditions (1 g). The experiments lasted 7 or 14 days. We report data on AT-MSCs proliferation as DNA content, and on the expression of specific osteogenic markers (cbfa-1, alkaline phosphatase activity and Van Kossa staining).

A NMR-compatible and reduced gravity simulation based (NRG) bioreactor for on-line monitoring cell culture metabolism.

We developed a NMR-compatible microgravity-based bioreactor (NRG[R]) that offers the advantage of an analytical non-invasive approach associated to the effects of an optimized suspension culture. The simulated microgravity conditions reached in the bioreactor are analogous to those of commercial apparatus like the Rotating Wall Vessel (RWV) system. The faster proliferation of endothelial cells cultured in the NRG bioreactor (doubling time : 28 +/- 1.7 vs. 43 +/- 5.6 h of the control grown in RWV) are attributed to different oxygenation conditions and medium wash out.

Pyridoneimines and pyridonemethides: substituent- and solvent-tunable intramolecular charge transfer and geometric isomerism.

We have prepared and fully characterized by means of multinuclear NMR and UV-vis spectroscopy a series of pyridoneimines and pyridonemethides in order to show how it is possible to finely tune pi-electron structure properties by properly exploiting substituent and solvent effects. Substituents with different electron-withdrawing capacities were introduced in pyridoneimines 2-4, pyridonemethides 5 and 6, and pyridine sulfonamido derivatives 7-9. The anisochrony of the carbon position of the azinium ring (geometric isomerism) and the exploitation of previously reported (13)C and (15)N shift/pi-electron density relationships allowed the investigation of the extent of intramolecular charge transfer from the donor group to the acceptor pyridinium moiety. By combining different substitutions with the polarity of the surrounding media, we were able to access a whole range of push-pull electron structures in solution, from fully aromatic-zwitterionic to quinoid-neutral, through many possible intermediate situations along the path. Due to the strict correlation between the pi-electron structure of push-pull derivatives and many photonic properties such as nonlinear optical activity, we believe that the achieved results should be valuable for the development of new efficient, tailor-made, heteroaromatic systems with optimized features as advanced organic materials.

Role of adenosine and glycogen in ischemic preconditioning of rat hearts.

We tested whether ischemic preconditioning of the rat heart is mediated by reduced glycogenolysis during ischemia, an event triggered by adenosine A1 receptor activation. Rat hearts (n=40) were studied with [31P] and [13C] nuclear magnetic resonance (NMR) spectroscopy, using the Langendorff perfusion technique (5.5 mM [1-13C]glucose, 10 U/l insulin). In parallel experiments, hearts (n=43) were freeze-clamped at different time-points throughout the protocol. They were subjected to either ischemic preconditioning (PC), PC in the presence of 50 microM adenosine receptor antagonist, 8-(p-sulfophenyl)-theophylline (SPT), or intermittent infusion of 0.25 microM adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA). After 30 min ischemia and reperfusion, recovery of heart ratexpressure product was improved in hearts treated with preconditioning (33+/-13%) or CCPA (58+/-14%) compared with the SPT and ischemic control (IC) groups, which both failed to recover (P<0.05). CCPA administration induced a 58% increase in pre-ischemic [13C]glycogen (P<0.05 vs. all groups). In the PC and SPT groups, [13C]glycogen decreased by 25 and 47%, respectively (P<0.05) due to the short bouts of ischemia, resulting in lower pre-ischemic glycogen compared to ischemic control and CCPA hearts (P<0.05). The rate of [13C]glycogen utilization during the first 15 min of ischemia (in micromol/min g wwt) was not statistically different between IC (0.42+/-0.03), PC (0.30+/-0.04), and CCPA (0.38+/-0.05) hearts, but was reduced in SPT hearts (0.24+/-0.05; P<0.05). Total glycogen depletion during 30-min ischemia was reduced in PC hearts (0.61 mg/g wwt) compared to IC (1.84 mg/g wwt) and CCPA (1.75 mg/g wwt) hearts; SPT did not block reduced glycogenolysis during ischemia in PC hearts (0.77 mg/g wwt vs. IC). This study adds further strong evidence that in rat hearts, adenosine is involved in ischemic preconditioning. However, protection is unrelated to pre-ischemic glycogen levels and glycogenolysis during ischemia.

Glycogen turnover and anaplerosis in preconditioned rat hearts.

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.

The role of adenosine in preconditioning.

Preconditioning is a powerful form of (myocardial) protection that follows brief sublethal ischemia. G-protein-coupled receptors constitute the trigger for entrance to the preconditioned state. In conjunction with other receptors, various membrane adenosine receptors play an important role in the transduction of extracellular signals, leading to protection by preconditioning, lasting 1-3 hr. Adenosine A(1)- and A(3)-receptors mediate inhibition of adenylate cyclase via a guanine nucleotide binding inhibitory protein (G(i/o)). A(2)-receptors couple to a comparable stimulatory protein (G(s)). Adenosine receptors are especially abundant in the central nervous system; in lesser numbers, they are found in many tissues, including the heart. A(1)-receptors are located on cardiomyocytes and vascular smooth muscle cells, A(2)-receptors on endothelial and vascular smooth muscle cells, and A(3)-receptors on ventricular myocytes. Ischemic preconditioning by endogenous adenosine takes place through A(1)- and A(3)-receptors. A(2A/B)-receptor activation results in vasodilation. The relevance of cellular mediators, such as 5'-nucleotidase, to generate adenosine for preconditioning is controversial. In contrast, the role of protein kinase C (PKC) is clearly established. Signals from different receptors converge at PKC, reaching a threshold activation of the kinase necessary to induce protection. Tyrosine and mitogen-activated protein kinases may play a role in addition to PKC. The exact products downstream responsible for the memory of preconditioning are elusive. A prime candidate for the end-effector of preconditioning is the K(ATP) channel. Preconditioning with adenosine-receptor agonists offers the possibility for treatment of coronary artery disease, but research in this field is still in its infancy.

Does resveratrol induce pharmacological preconditioning?

Resveratrol is a grape component with complex pharmacology related to its antioxidant activity. Little is known about the direct effects of resveratrol on the myocardium. We tested whether resveratrol administration before ischemia could attenuate ischemic/reperfusion damage. We examined how resveratrol affects high-energy phosphate metabolism (31P-nuclear magnetic resonance) and contractility of isolated Langendorff perfused rat hearts subjected to 20 min no-flow ischemia and 30 min reperfusion. During 10 min resveratrol infusion (10 microM) before ischemia, basal phosphorylation potential dropped by 40% (p < 0.05 vs. preinfusion value) without affecting contractility. The level of effluent adenosine was increased by 68%, parallel to a 50% increase in coronary flow. Resveratrol significantly improved postischemic recovery of rate-pressure product (62 +/- 5.2 vs. 23 +/- 8.1% of controls; p < 0.05). The metabolic pattern following resveratrol infusion was similar to that produced by ischemic preconditioning, suggesting that an increase in adenosine availability is involved in cardioprotection.

Carbohydrates and purines in underperfused hearts, protected by ischemic preconditioning.

Few results, and those controversial, have been published on ischemic preconditioning followed by low-flow ischemia. The aim of this study was to assess whether ischemic preconditioning: (1) confers protection against severe underperfusion; and (2) is mediated by mobilization of proglycogen, resulting in increased anaerobic glycolysis and reduced myocardial injury. Isolated rat hearts were retrogradely perfused and subjected to either 25 min low-flow ischemia (0.6 ml/min) followed by 30 min reperfusion (IC; n=5), or the same protocol preceded by two cycles of 5 min no-flow ischemia and 5 min reperfusion (PC; n=7). Additionally, hearts (n=52) were freeze-clamped at different time points throughout the protocol. Preconditioning improved functional recovery (developed force X heart rate in PC hearts: 54 v 21% in IC hearts; P<0.01) and reduced ischemic damage (cumulative release of creatine kinase during reperfusion: 93 v 215 micro/g dry weight; P<0.05). During ischemia and reperfusion, release of adenosine and the sum of purines was smaller in PC hearts (P<0.05), while lactate release was similar in the two groups. PC reduced both macroglycogen and proglycogen by c. 60% (P<0.01) resulting in constant glycogen levels during low-flow ischemia. In contrast, in IC hearts, both fractions decreased by c. 60% during underperfusion (P<0.01). These results demonstrate that: (1) ischemic preconditioning reduces injury due to severe flow reduction; and (2) preconditioning reduced glycogenolysis without affecting anaerobic glycolysis, suggesting increased glucose uptake.

Controversies in preconditioning.

Preconditioning is an effective mean of protecting the heart against prolonged ischemia by pretreating it with a minor insult, and the present paper reviews various controversies in this highly active field of research. In many models, adenosine plays a role by triggering the activation of protein kinase C. It may work in conjunction with other agents, such as bradykinin, but the putative role of noradrenaline is uncertain. Regulation of the enzyme producing adenosine (i.e., 5'-nucleotidase) has been reported during preconditioning but, because its activity does not seem to be associated with infarct size, it is unlikely that the hydrolase plays a pivotal role. Controversial data have been published on the involvement of mitochondrial ATPase, which may be ascribed to the poor time resolution of the experiments described; however, we do not believe that either acidosis or tissue ATP are important factors in triggering preconditioning. The role of glycolysis in the preconditioning effect remains to be firmly established; opposite mechanisms are activated in low-flow and stop-flow protocols. Although species differences regarding preconditioning exist, they seem to be more of a quantitative than a qualitative nature. The phenomenon could be clinically relevant because evidence is accumulating that preconditioning may take place during bypass surgery and coronary angioplasty if longer balloon-occlusion times are used.

Induction of human endothelial cell growth by mildly oxidized low density lipoprotein.

The aim of the present study was to examine whether endothelial growth could be modulated by mildly oxidized low density lipoprotein. When human endothelial cells were cultured in the presence of mildly oxidized low density lipoprotein (1 microgram/ml), a significant induction of endothelial cell growth was observed, whereas native low or high density lipoprotein were ineffective. Further, treatment of endothelial cells with mildly oxidized low density lipoprotein modulated the expression of cytokines and growth factors which may be relevant in atherogenesis. Endothelial cells chronically exposed to mildly oxidized low density lipoprotein underwent a more rapid onset of cellular senescence. Since senescence is associated with endothelial dysfunction, the novel finding showing that mildly oxidized low density lipoprotein induces endothelial cell growth may be relevant in the development and evolution of the atherosclerotic lesions.

Pharmacological action of a new spin trapping compound, 2-phenyl DMPO, in the adriamycin-induced cardiotoxicity.

Adriamycin (ADR)-induced cardiotoxicity was adopted in this investigation as a reliable model of radical-dependent myocardial pathology allowing both quantitative studies of drug activity in the isolated organ and in vivo comparison of the cardio-protection vs. general toxicity. Since commercially available lipophilic spin trapping compounds were shown to develop significant protective activity, in this investigation a newly synthesized spin trap (2-phenyl-DMPO) was studied. In Langendorff rat heart, 200 microM ADR induced a significant impairment of contractile performance, while 2-phenyl-DMPO was not cardiotoxic up to the 5 mM concentration. By this dose, 2-phenyl-DMPO induced a significant protection against the ADR-induced contractile impairment. In in vivo experiments, ADR (9 mg/kg i.v.) produced a significant impairment of ECG, coronary flow and contractility. The continuous administration of 2-phenyl-DMPO i.p. by osmotic pump delivering 0.3 mumol/hr was unable to protect the animals against the cardiotoxic signs. Seven days after ADR administration, severe general toxicity (arrest of body weight increase) and myelotoxicity were also observed. 2-phenyl-DMPO was unable to protect the animals from these toxic signs. The present results confirm that lipophilic spin traps can be a new class of antiradical drugs, as confirmed by the experiments performed in the isolated heart with the 2-phenyl-DMPO; however, this last compound is probably metabolized in vivo to inactivate derivatives.

NMR evaluation of changes in myocardial high energy metabolism produced by repeated short periods of ischemia.

Following our previous results which demonstrated that repeated short periods (2 min) of ischemia are capable of protecting the isolated rat heart from a subsequent global ischemia (30 min), in the present study we have concentrated on the metabolic changes occurring in rat hearts during six 2 min ischemia/3 min reperfusion cycles. Cardiac high-energy phosphates were monitored using 31P-NMR. Phosphocreatine levels fell (50-60%) during each ischemic period, and recovered to 70-80% of their initial values during reperfusion. P(i) rose by 59% during the first ischemic period, but increased less during subsequent ischemias (30% during the 6th occlusion, P < 0.05 vs. the first ischemic period) returning to baseline levels after each reperfusion. [ATP], pH, and [Mg2+] remained almost unaffected, but there was a decrease in HPLC-determined effluent ATP catabolites. The first occlusion led to a 95% drop in contractile function (P < 0.001 vs. baseline), but this recovered to 73% upon reperfusion (P < 0.02 vs. baseline), and was 65% at the end of the protocol. Phosphorylation potential (PP = [ATP]/([ADP].[P(i)]) correlated exponentially with total purine (r = 0.90) and with adenosine + inosine release (r = 0.81), and by the 6th ischemia/reperfusion cycle, exceeded that observed in controls by 21% (P < 0.05). We conclude that repeated short periods of ischemia do not lead to any significant alteration in the absolute myocardial ATP, but are associated with an enhanced cytosolic energy state in the heart, that enables the myocardium to reach a steady albeit lower functional state. Adenosine (+inosine) release may be involved in the regulation of the energy supply-demand balance.

Intermittent v continuous ischemia decelerates adenylate breakdown and prevents norepinephrine release in reperfused rabbit heart.

Myocardium tolerates intermittent ischemia followed by short reperfusions better than continuous ischemia of the same duration. We attempted to delineate the differential mechanism(s) involved in intermittent v continuous ischemia. Isolated, paced rabbit hearts were perfused at 22 ml/min. Coronary flow was stopped intermittently 12 x for 2 or 4 min, with 3-min reperfusions (total reperfusion period: 36 min). In two other groups, flow was stopped continuously for 24 or 36 min followed by a flat 36-min reperfusion. Following the first intermittent 2-min ischemia, adenosine efflux increased ninefold; in all subsequent ischemia/reperfusion cycles, adenosine and total purine releases were substantially less despite identical heart rates, coronary flows and ischemic periods. The rate-pressure product prior to the intermittent ischemias exhibited exponential correlations with total purine efflux during the 3 min of reperfusion. When intermittent ischemia was extended to 4 min, the initial attenuation of ATP breakdown during the prior 2-min occlusions was overcome, but during subsequent 4-min ischemia/reperfusion cycles, ATP breakdown was again attenuated relative to the first 4-min ischemia. After the prolonged continuous ischemias, purine efflux was up to 6 x higher than with intermittent ischemias of the same total time of zero flow. Lactate release and hence cellular H+ export after intermittent ischemias remained consistently elevated, but following the continuous ischemia of 36 min, release of lactate, and thus H+, was subsequentially decreased. Glycogen mobilization occurred regardless of the ischemia's nature, but it was markedly enhanced during continuous ischemias, where no fall in proglycogen levels occurred. Similarly, myocardial norepinephrine release increased substantially only during the prolonged continuous ischemias. Thus short intermittent ischemia attenuates cardiac adenylate degradation and glycogen mobilization; such ischemic insult also provides for better lactate and H+ washouts immediately upon reperfusion. Another beneficial effect of intermittent ischemia was the near-complete absence of free interstitial norepinephrine, which exacerbates myocardial ischemic insults. In addition, the exponential correlations between preischemic rate-pressure product and postischemic purine release suggest that preischemic energy demand may determine ATP breakdown in ischemic rabbit myocardium.

Synthesis and free radical scavenging properties of the enantiomers of erdosteine.

The synthesis of enantiomers R and S of erdosteine, a derivative of homocysteine-gamma-thiolactone, and the NMR studies for the determination of the enantiomeric excess with chiral shift reagent on the more soluble ethyl esters, are described. Pharmacological data relative to the free radical scavenging properties of the R and S enantiomers are reported. In particular, it has been documented that the S isomer is more effective than R isomer in protecting mice against lethal doses of paraquat (substance able to form free radicals when administered by i.p. route).

Modulators of oxidized LDL-induced hyperadhesiveness in human endothelial cells.

The adherence of monocytes to the endothelium is an early event in atherogenesis. Our previous studies have demonstrated that oxidized LDL induced U937 cells-endothelial interactions and that HDL prevented oxidized LDL effects. Here, we provide evidence that treatment of endothelial cells with the anti-inflammatory agent indomethacin abolished oxidized LDL as well as interleukin 1- and lipopolysaccharide-stimulated U937 adhesion. It is noteworthy that HDL, which is known to be protective against atherosclerosis, was effective only in negating U937 adhesion induced by oxidized LDL, while it did not affect interleukin 1- and lipopolysaccharide-induced hyperadhesiveness in endothelial cells. Since indomethacin inhibits cyclooxygenase which is the key enzyme in the synthesis of prostanoids, we have studied the effect of oxidized LDL on the expression of cyclooxygenase type 2 and demonstrated that oxidized LDL induces a sustained increase in the expression of cyclooxygenase mRNA.

The protective role of high-density lipoprotein on oxidized-low-density-lipoprotein-induced U937/endothelial cell interactions.

The adherence of monocytes to the endothelium is an early event in atherogenesis. We have investigated this process by examining whether native and oxidized low-density and high-density lipoproteins could modulate this process. Only oxidized low-density lipoprotein caused a significant dose-dependent and time-dependent increase in U937 monocyte-like cell line binding to human endothelial cells, by a process which required de novo protein synthesis. Interestingly, E-selectin, intercellular adhesion molecule-1, vascular cell-adhesion molecule or P-selectin induction was not apparent in this system suggesting the presence of an alternative system for the interaction of endothelial cells with monocyte-like cells in response to oxidized low-density lipoprotein. High-density lipoprotein completely suppressed oxidized low-density-lipoprotein-induced adhesion of U937 cells to the endothelial monolayer, while oxidized high-density lipoprotein did not. These data suggest that the balance between native and oxidized lipoproteins may play a role in the formation of the atherosclerotic lesion by modulating monocyte endothelial interactions.

Intracellular magnesium homeostasis is involved in the functional recovery of preconditioned rat heart.

The role of ionic derangements in preconditioned hearts (PC) was assessed by measuring pHi, high-energy phosphates (HEP) and [Mg2+] by 31P NMR. Control (C) Langendorff rat hearts were subjected to 30' ischemia and 30' reflow. PC underwent 4x(2' ischemia + 3' reperfusion) before prolonged ischemia and reflow. In this model, the contractile recovery of PC hearts at the end of reflow (rate-pressure product RPP: 50 +/- 12% vs. 5 +/- 5% of C, p = 0.004) is not related to higher HEP or pHi levels than in C. [Mg2+]i increased significantly during ischemia both in C and PC organs; upon reperfusion its level remained significantly high in C (p < 0.001), while it regained the normal value in PC hearts. This behavior might improve in turn the SR calcium handling in PC organs, eventually contributing to the contractile recovery.

The hydrophilic spin trap, 5,5-dimethyl-1-pyrroline-1-oxide, does not protect the rat heart from reperfusion injury.

The role and site of free radical generation in myocardial ischemia/reperfusion injury were investigated using the hydrophilic spin trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). DMPO (40 mM) proved ineffective in preserving the contractile performance and energy metabolism of Langendorff-perfused rat hearts following ischemia and reperfusion. This result, which is in contrast with the cardioprotection observed with hydrophobic spin traps, suggests that free radicals are generated intracellularly under these conditions.