Effects of acute versus post-acute systemic delivery of neural progenitor cells on neurological recovery and brain remodeling after focal cerebral ischemia in mice.

Intravenous transplantation of neural progenitor cells (NPCs) induces functional recovery after stroke, albeit grafted cells are not integrated into residing neural networks. However, a systematic analysis of intravenous NPC delivery at acute and post-acute time points and their long-term consequences does not exist. Male C57BL6 mice were exposed to cerebral ischemia, and NPCs were intravenously grafted on day 0, on day 1 or on day 28. Animals were allowed to survive for up to 84 days. Mice and tissues were used for immunohistochemical analysis, flow cytometry, ELISA and behavioral tests. Density of grafted NPCs within the ischemic hemisphere was increased when cells were transplanted on day 28 as compared with transplantation on days 0 or 1. Likewise, transplantation on day 28 yielded enhanced neuronal differentiation rates of grafted cells. Post-ischemic brain injury, however, was only reduced when NPCs were grafted at acute time points. On the contrary, reduced post-ischemic functional deficits due to NPC delivery were independent of transplantation paradigms. NPC-induced neuroprotection after acute cell delivery was due to stabilization of the blood-brain barrier (BBB), reduction in microglial activation and modulation of both peripheral and central immune responses. On the other hand, post-acute NPC transplantation stimulated post-ischemic regeneration via enhanced angioneurogenesis and increased axonal plasticity. Acute NPC delivery yields long-term neuroprotection via enhanced BBB integrity and modulation of post-ischemic immune responses, whereas post-acute NPC delivery increases post-ischemic angioneurogenesis and axonal plasticity. Post-ischemic functional recovery, however, is independent of NPC delivery timing, which offers a broad therapeutic time window for stroke treatment.

Cellular effects of factor Xa on vascular smooth muscle cells--inhibition by heparins?

In addition to its key function as a clotting enzyme, factor Xa (FXa) also elicits cellular effects. In cultured human venous smooth muscle cells (SMCs), FXa induced a mitogenic response that was independent of thrombin and platelet-derived growth factor (PDGF). Unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) (enoxaparin) inhibited the mitogenic effects of FXa, thrombin and fetal calf serum (FCS), but did not reduce mitogenesis induced by PDGF. Similarly, both UFH and LMWH inhibited the activation of extracellular signal-regulated kinase (ERK-1/2) by FXa, thrombin and FCS, but not by PDGF. This indicates that heparins can influence cellular signaling in SMC via an antithrombin-II (AT-III)-independent mechanism. The inhibition of ERK-1/2 correlated with the inhibition of mitogenesis by the heparins. Thus, the inhibition of ERK-1/2 phosphorylation by heparins might predict an antimitogenai response in this system.

Evidence for functionally active protease-activated receptor-4 (PAR-4) in human vascular smooth muscle cells.

1. This study investigates, whether in addition to the protease-activated receptor-1 (PAR-1), PAR-4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR-1 and PAR-4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. 2. mRNAs for both, PAR-1 and PAR-4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT - PCR). 3. Treatment of the SMC with GYPGQV (200 microM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. 4. Stimulation of the SMC with 10 nM thrombin or 200 microM SFLLRN caused a time-dependent activation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 microM of GYPGQV induced a prolonged phosphorylation of ERK-1/2 with a maximum at 60 min. These data suggest that PAR-1 and PAR-4 are activated by thrombin at distinct concentrations and with distinct kinetics. 5. GYPGQV stimulated [(3)H]-thymidine incorporation in SMC. At 500 microM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 microM SFLLRN, respectively. 6. These data indicate that a functionally active PAR-4 is present in SMC and, in addition to PAR-1, might contribute to thrombin-induced mitogenesis.

Factor Xa acts as a PDGF-independent mitogen in human vascular smooth muscle cells.

This study investigates the mitogenic effect of the coagulation factor Xa in smooth muscle cells (SMC) from human saphenous vein and the procoagulant activity of these cells. Factor Xa elicited a concentration-dependent increase in [3H]thymidine incorporation. This mitogenic effect of factor Xa was inhibited by DX-9065a and BABCH, indicating the requirement of proteolytic activity of the enzyme. Factor Xa activated the MAP kinases ERK1/2 concentration- and time-dependently. PDGF-neutralizing antibodies neither inhibited the increase in [3H]thymidine incorporation nor ERK-1/2 phosphorylation in factor Xa-stimulated cells, suggesting that factor Xa-induced signaling and mitogenic activity in human venous SMC are independent of PDGF. Exposure of SMC to recalcified plasma resulted in a significant thrombin generation which was inhibited by anti-tissue factor antibody, tissue factor pathway inhibitor, inactivated factor VIIa and DX-9065a. These data indicate that interaction of SMC with the clotting system may contribute to venous graft disease, i.e. thrombus formation and intimal hyperplasia.

Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells.

1. This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinase-activated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar effect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. 3. The specific PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5. Trypsin (2 nM) as well as SLIGRL (100 microm) activated the nuclear factor KB (NFkappaB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkappaB by thrombin (10 nM) was detected after 4-5 h. 6. These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkappaB via either PAR-1 or PAR-2 does not predict mitogenesis.

Thrombin-induced mitogenesis in coronary artery smooth muscle cells is potentiated by thromboxane A2 and involves upregulation of thromboxane receptor mRNA.

BACKGROUND: Previous studies have shown that thrombin is a potent though slow-acting mitogen for vascular smooth muscle cells (SMC). Because thrombin generation in vivo is accompanied by platelet activation, it has been suggested that platelet-derived factors might enhance thrombin-induced SMC proliferation. No information is available so far on the possible role of thromboxane A2. METHODS AND RESULTS: Thrombin (1 U/mL) caused a threefold to fourfold increase of DNA synthesis in cultured bovine coronary artery SMC as assessed from [3H]thymidine incorporation. U 46619, a stable thromboxane A2 mimetic, had only a minor stimulating effect on its own but potentiated the thrombin effect sixfold to sevenfold above control (P<.05). These findings were paralleled by a 52+/-5% (P<.05) increase in cell number at 48 hours after addition of both mitogens as compared with 24+/-5% with thrombin alone and no change with U 46619 alone. Thromboxane A2 receptor mRNA was found to be upregulated sixfold 20 minutes after thrombin stimulation. Pretreatment of SMC with thrombin for 4 hours markedly increased U 46619-induced mitogen-activated protein kinase activity, indicating thrombin-induced upregulation of functional thromboxane receptors in SMC. CONCLUSIONS: Thrombin-induced proliferation of SMC is markedly enhanced by thromboxane A2. This might result in an enhancement of SMC proliferation by platelet-derived thromboxane A2 in vivo.

Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects.

Activation of NFkappaB is essential but not sufficient to stimulate mitogenesis of vascular smooth muscle cells.

This study investigates the role of the transcription factor NFkappaB in thrombin- and thrombin receptor activating peptide (TRAP, SFLLRNPNDKYEPYF)-induced mitogenesis of cultured bovine coronary artery smooth muscle cells (SMC). Stimulation of resting cells by thrombin (10 nM) or TRAP (10-100 microM) resulted in a comparable time-dependent activation of NFkappaB as detected by Western blotting and electrophoretic mobility shift assay (EMSA) of nuclear extracts. The NFkappaB activation was antagonized by N-acetyl-L-cysteine (20 mM) and pentoxifylline (0.5 mM). Thrombin caused a 3-4-fold increase in [3H]thymidine incorporation within 24 h which was prevented by inhibitors of NFkappaB activation. In contrast, TRAP did not cause any mitogenic response. These results demonstrate that activation of NFkappaB is an essential but not a sufficient signal for SMC mitogenesis.

Thromboxane A2 potentiates thrombin-induced proliferation of coronary artery smooth muscle cells.

The activation of thrombin is the key event in clot formation after vascular injury. Thrombin itself, but also other clot-derived factors, such as thromboxane A2 (TXA2), are mitogenic for vascular smooth muscle cells. We have studied the possible interactions between thrombin and TXA2 in stimulation of coronary artery smooth muscle cell (SMC) proliferation. Thrombin (1 U/ml) caused a significant proliferatory response in SMC. U 46619, a stable TXA2 mimetic, had only a minor stimulating effect by its own but markedly potentiated the thrombin-induced mitogenesis. A possible mechanism for these potentiating effects is provided by the demonstration of a marked (6 fold) but transient (maximum after 20 min) increase in the expression of TXA2 receptor (TP receptor) mRNA in SMC by thrombin. Since a significant clot-related TXA2 generation was detected for at least 2 hours, the up-regulation of TP receptors by thrombin may represent a mechanism that is relevant for the in vivo situation of SMC proliferation after vessel injury.

Relaxant and contractile responses of porcine pulmonary arteries to thrombin and thrombin receptor activating peptides.

The vascular effects of thrombin and thrombin receptor activating peptides (TRAP) were studied on isolated rings from porcine pulmonary arteries. In prostaglandin F2 alpha (PGF2 alpha)-precontracted vessels with intact endothelium, both thrombin- and TRAP-induced nitric oxide-mediated relaxation, whereas in endothelium-denuded vessels thrombin and TRAP elicited concentration-dependent contractile responses. The first phasic component of contraction was associated with increased generation of inositol 1,4,5-triphosphate and the tonic component seemed to be due to the activation of protein kinase C. Both peptides (TRAP-6 with 6 and TRAP-14 with 14 amino acid residues) did not differ in their intrinsic activity; like thrombin, both peptides elicited dualistic vascular effects but their potency was more than three orders of magnitude less than that of thrombin.

Inositol 1,4,5-triphosphate and protein kinase C are involved in thrombin- and trap-induced vascular smooth muscle contraction.

Thrombin (30 nmol/l) as well as the thrombin receptor activating peptide (TRAP), 10 mumol/l) induce a sustained contraction of endothelium-denuded porcine pulmonary arteries. The first phasic component of contraction is associated with the generation of IP3 which precedes the development of contractile force. Since the PKC inhibitor staurosporine (50 nmol/l) completely inhibits the tonic contraction this component of contraction seems to be due to the activation of protein kinase C (PKC). The thrombin- and TRAP-induced vasoconstriction strongly depends on extracellular calcium; the remaining thrombin- or TRAP-induced contraction in Ca(2+)-free medium seems to be attributed to the IP3-mediated release of calcium from intracellular stores.

Involvement of inositol 1,4,5-triphosphate and protein kinase C in thrombin-induced contraction of porcine pulmonary artery.

The role of the intracellular messengers inositol 1,4,5-triphosphate (IP3) and protein kinase C (PKC) in the thrombin (3 U/mL)-induced contraction of endothelium-denuded porcine pulmonary arteries was investigated. Thrombin induced a sustained contractile response with an initial transient increase in IP3 to about 160% of the unstimulated control. Omission of extracellular Ca2+ or preincubation with verapamil (10 mumol/L) reduced the maximum of contraction without significantly affecting the thrombin-induced increase in IP3. To evaluate the role of PKC for the contractile response, the PKC was activated directly by phorbol 12,13-dibutyrate (PDBu, 50 nmol/L). The phorbol ester produced a slowly increasing tonic contraction without any changes in the basal IP3 level. There was a moderate inhibition of PDBu-induced contractions in Ca(2+)-free solution, while they were not inhibited after preincubation with verapamil. Preincubation with the PKC inhibitor staurosporine (50 nmol/L) significantly reduced the PDBu-induced contraction (by about 80%). In thrombin-stimulated vessels staurosporine only inhibited the tonic phase of the contractile response whereas the increase in IP3 and the phasic component of contraction were still evident. These results suggest that IP3 and PKC are involved in the thrombin-induced contraction. The phasic component of contraction is associated with the generation of IP3; the tonic component might be due to the activation of PKC.

ADP-, PAF- and adrenaline-induced platelet aggregation and thromboxane formation are not affected by a thromboxane receptor antagonist at physiological external Ca++ concentrations.

Thromboxane (TX) receptor antagonists are of considerable clinical interest in prevention of acute thrombembolic vessel occlusion. This study demonstrates that the selective TX receptor antagonist, daltroban, at a concentration (10 microM) that does not inhibit TX synthesis, markedly inhibits ADP-, PAF- and adrenaline-induced platelet secretion and TX formation. With the exception of ADP-induced platelet secretion, these actions are only detectable in citrated platelet-rich plasma but not in plasma anticoagulated by hirudin. Since TX antagonists are supposed to act at physiological external Ca++ concentrations in the clinics, it is questionable whether in vitro studies in Ca(++)-deprived media are the optimum model to evaluate the clinical potential of these compounds.

Contractile effects of thrombin in porcine pulmonary arteries and the influence of thrombin inhibitors.

Thrombin catalyzes not only the conversion of fibrinogen to fibrin but also activates several receptor-mediated cell responses. In ring segments of porcine pulmonary arteries the contractile effect of thrombin was studied in the presence and absence of endothelium. The integrity of endothelium was assessed by the bradykinin-induced relaxation of PGF2 alpha (3 mumol/l)-precontracted vessels which was absent after mechanical removal of endothelium. Thrombin at 0.1 to 10 U/ml (i.e. about 1-100 nmol/l) caused a sustained contraction in endothelium-denuded arteries with a maximum at 20-30 min. In vessels with intact endothelium a significant increase in tension up to 1 U/ml was observed preceded by a transient relaxant response. The contractile effect in vessels with intact endothelium was comparatively weaker. This is probably due to the release of EDRF from endothelial cells since blockade of EDRF synthesis by NG-nitro-L-arginine augmented the thrombin-induced contractions in arteries with intact endothelium. Indomethacin did not alter the contractile effect. However, in vessels with endothelium and in endothelium-denuded vessels the contractions were reduced when extracellular calcium was omitted. Verapamil (10 mumol/l) significantly diminished the contractile effect only in endothelium-denuded vessels. On preincubation of endothelium-denuded arterial ring segments with myo-[2-3H]inositol the addition of thrombin (10 U/ml) caused an accumulation of [3H]inositol-1,4,5-triphosphate (IP3). A maximum was observed after 2 min preceding the maximum increase in contraction. Measurement of thrombin-induced endogenous IP3 generation by radioreceptor assay yielded the same results. The thrombin-induced contractile effect requires the proteolytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)