[Layer-specific strain assessment on left ventricular function before and after PCI in patients with ST segment elevation myocardial infarction].

Objective: To evaluate the changes of left ventricular function in patients with ST segment elevation myocardial infarction (STEMI) before PCI and within 24 hours after PCI by layer-specific strain, and to explore the value of this new assessment method for quantitative monitoring on the myocardial function in STEMI patients. Methods: A total of 40 patients with acute anterior wall myocardial infarction, who underwent PCI in Affiliated Hospital of Jiangsu University during July 2017 to July 2018, were included in this prospective cohort study. According to the symptom to balloon time (STB), the patients were divided into STB ≤6 hours group (26 cases) and STB 6-12 hours group (14 cases). Echocardiography was performed before, immediately, 3 hours and 24 hours after PCI. Echocardiographic indexes including endocardial myocardial longitudinal strain (LS-endo), 18-segment full-thickness myocardial longitudinal strain (LS) of left ventricle and left ventricular global longitudinal strain (GLS) were measured. The mean LS-endo and LS values of myocardial segments in infarcted area (IALS-endo, IALS) and the mean LS-endo and LS values of myocardial segments in non-infarcted area (NIALS-endo, NIALS) were calculated. Results: There were 34 males and 6 females in this cohort and age was (62±10) years. In STB≤6 hours group, the IALS-endo value ((13.7±4.9)% vs. (10.0±2.7)%, P<0.05) and NIALS-endo value ((17.0±2.9)% vs. (14.6±2.9)%, P<0.05) were significantly higher at 24 hours after PCI than those before PCI. In the group of STB 6-12 hours, IALS-endo decreased immediately after PCI ((6.7±3.3)% vs. (11.9±6.5)%, P<0.05), and there was a rising trend at 3 hours after PCI (P>0.05). At 24 hours after PCI, the index was higher than that immediately after PCI ((13.6±8.4)% vs. (6.7±3.3)%, P<0.05). The NIALS-endo value was significantly higher at 24 hours after PCI than that before PCI ((17.1±2.1)% vs. (14.5±3.2)%, P<0.05). In the STB 6-12 hours group, the decrease rate of IALS-endo immediately after PCI was higher than that in the STB ≤6 hours group (93% (13/14) vs. 35% (9/26), P<0.001). In STB ≤6 hours group, the NIALS value at 24 hours after PCI was higher than that before PCI (P<0.05), and there was no significant difference in IALS, NIALS and GLS at other time points (P>0.05). Conclusions: Layered LS is superior to full-thickness LS and GLS in evaluating left ventricular function in STEMI patients. LS measured by echocardiography can continuously and quantitatively evaluate the changes of left ventricular myocardial function in STEMI patients before and after PCI.

Reducing 3,4-dihydroxyphenylpyruvic acid to d-3,4-dihydroxyphenyllactic acid via a coenzyme nonspecific d-lactate dehydrogenase from Lactobacillus reuteri.

AIMS: The purpose of this work was to find an efficient enzyme to synthesize d-3,4-dihydroxyphenyllactic acid (d-DSS). METHODS AND RESULTS: Nineteen lactic acid bacteria strains were screened for production of d-DSS using 3,4-dihydroxyphenylpyruvic acid (DPA) as a substrate. Lactobacillus reuteri JN516 exhibited the highest d-DSS yield. A nonspecific coenzyme, d-lactate dehydrogenase (d-LDH82319), from L. reuteri JN516 with high DPA reducing activity was identified. This enzyme reduced DPA to form d-DSS with excellent optical purity (enantioselectivity >99%). Its molecular weight was 35 kDa based on SDS-PAGE migration. The Michaelis-Menten constant (Km ), turnover number (kcat ), and catalytic efficiency (kcat /Km ) of d-LDH82319 for DPA were 0·09 mmol l-1 , 2·17 s-1 and 24·07 (mmol l-1 )-1  s-1 , respectively, with NADH as the coenzyme. The (Km ), (kcat ) and (kcat /Km ) of d-LDH82319 for DPA were 0·10 mmol l-1 , 0·13 s-1 and 1·30 (mmol l-1 )-1  s-1 , respectively, with NADPH as the coenzyme. The optimum temperature and pH of d-LDH82319 were 25°C and pH 8 respectively. Additionally, d-LDH82319 had a broad substrate range for alpha-keto acids, among which the activity of reducing pyruvate was the strongest; therefore, it belongs to the group of d-lactate dehydrogenases. d-LDH82319 and glucose dehydrogenase (GDH) were coexpressed to produce d-DSS from DPA. CONCLUSIONS: d-LDH82319 from L. reuteri JN516 with high DPA reducing activity has the characteristics of a nonspecific coenzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: d-LDH82319 is the first reported coenzyme nonspecific d-lactate dehydrogenase with DPA-reducing activity. The coexpression system provided an effective method to produce d-DSS.

Angiomodulatory and neurological effects of ginsenosides.

Panax ginseng C.A. Meyer, one of the most popular and valued herbs, has been used extensively in traditional Chinese medicine for thousands of years. More than thirty ginsenosides, the pharmacologically active ingredients in ginseng, have been identified with various sugar moieties attached at the C-3, C-6 and C-20 positions of the steroidal skeleton. We herein review the current literature on the pharmacological effects of ginsenosides on the modulation of angiogenesis, dysregulations of which contribute towards many pathological conditions. Regarding the adaptogenic property of ginseng, the effects of ginsenosides on central nervous system are also discussed. Recent researches have pointed to the steroid hormone receptors as the target molecules to elicit the diverse cellular and physiological activities of ginseng. We believe that understanding the interaction between ginsenosides and various steroid hormone receptors may provide clues to unravel the secret of ginseng.

Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity.

Ginsenoside-Rg1, the pharmacologically active component isolated from ginseng, demonstrated neuroprotective effects on primary cultured rat nigral neurons against rotenone toxicity. Rotenone, a common household pesticide known for its specific and irreversible mitochondria complex I inhibition, has been suggested to be the causal agent of Parkinson's disease (PD) by inducing degeneration of cells in the substantial nigra. The present study demonstrated that co-treatment of rotenone and Rg1 could reduce rotenone-induced cell death by 58% (SEM=+/-5.60; N=3). Rotenone-induced mitochondria membrane potential (MMP, DeltaPsim) depletion was restored and elevated by at least 38% (SEM=+/-2.15; N=3) by Rg1. In addition, Rg1 prevented cytochrome c release from the mitochrondrial membrane and increased the phosphorylation inhibition of the pro-apoptotic protein Bad through activation of the PI3K/Akt pathway. The protective effects of Rg1 was blocked by glucocorticoid receptor antagonist RU486, indicating that the action of Rg1 is mediated through glucocorticoid receptor (GR). In conclusion, Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of the primary cultured nigral neurons against rotenone toxicity. Thus, Rg1 and its related compounds may be developed as protective agents against neurodegenerative diseases induced by mitochondrial toxins.

Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen beta receptor.

BACKGROUND AND PURPOSE: Angiogenesis is a crucial step in tumour growth and metastasis. Ginsenoside-Rb1 (Rb1), the major active constituent of ginseng, potently inhibits angiogenesis in vivo and in vitro. However, the underlying mechanism remains unknown. We hypothesized that the potent anti-angiogenic protein, pigment epithelium-derived factor (PEDF), is involved in regulating the anti-angiogenic effects of Rb1. EXPERIMENTAL APPROACHES: Rb1-induced PEDF was determined by real-time PCR and western blot analysis. The anti-angiogenic effects of Rb1 were demonstrated using endothelial cell tube formation assay. Competitive ligand-binding and reporter gene assays were employed to indicate the interaction between Rb1 and the oestrogen receptor (ER). KEY RESULTS: Rb1 significantly increased the transcription, protein expression and secretion of PEDF. Targeted inhibition of PEDF completely prevented Rb1-induced inhibition of endothelial tube formation, suggesting that the anti-angiogenic effect of Rb1 was PEDF specific. Interestingly, the activation of PEDF occurred via a genomic pathway of ERbeta. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERbeta, but not ERalpha. Rb1 effectively recruited transcriptional activators and activated an oestrogen-responsive reporter gene. Furthermore, Rb1-mediated PEDF activation and the subsequent inhibition of tube formation were blocked by the ER antagonist ICI 182,780 or transfection of ERbeta siRNA, indicating ERbeta dependence. CONCLUSIONS AND IMPLICATIONS: Here we show for the first time that the Rb1 suppressed the formation of endothelial tube-like structures through modulation of PEDF via ERbeta. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy.

Signaling pathway of ginsenoside-Rg1 leading to nitric oxide production in endothelial cells.

We here provide definitive evidence that ginsenoside-Rg1, the pharmacologically active component of ginseng, is a functional ligand of the glucocorticoid receptor (GR) as determined by fluorescence polarization assay. Rg1 increased the phosphorylation of GR, phosphatidylinositol-3 kinase (PI3K), Akt/PKB and endothelial nitric oxide synthase (eNOS) leading to increase nitric oxide (NO) production in human umbilical vein endothelial cell. Rg1-induced eNOS phosphorylation and NO production were significantly reduced by RU486, LY294,002, or SH-6. Also, knockdown of GR completely eliminated the Rg1-induced NO production. This study revealed that Rg1 can indeed serve as an agonist ligand for GR and the activated GR can induce rapid NO production from eNOS via the non-transcriptional PI3K/Akt pathway.

The angiosuppressive effects of 20(R)- ginsenoside Rg3.

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.

Controlling the vasculature: angiogenesis, anti-angiogenesis and vascular targeting of gene therapy.

Angiogenesis is the development of new blood vessels from an existing vascular bed. Normal vascular proliferation occurs only during embryonic development, the female reproductive cycle and wound repair. By contrast, many pathological conditions (for example, cancer, atherosclerosis and diabetic retinopathy), are characterized by persistent, unregulated angiogenesis. Conversely, inadequate angiogenesis can lead to failure of ulcers to heal and myocardial infarction. Control of vascular development could permit new therapeutic approaches to these disorders. For example, several anti-angiogenic drugs are currently undergoing clinical trials for the treatment of cancer, whereas enhancement of angiogenesis by exogenous growth factors can prevent or limit the damage in chronic wounds and duodenal ulcers. Here Tai-Ping Fan, Rhys Jaggar and Roy Bicknell highlight recent achievements and discuss the prospects of receptor antagonists, enzyme inhibitors, tumour suppressor genes and vascular targeted approaches, especially that of gene therapy, in the future development of angiotherapy.

Mechanism of action of cyclosporine in preventing cardiac allograft rejection. II. Graft tissue levels of prostacyclin and thromboxane.

Four cyclo-oxygenase products (COP) of arachidonate were measured in tissue homogenates of rat cardiac allografts at intervals after transplantation. In rejecting graft tissue, there was a progressive decrease in the levels of prostaglandin E2 (PGE2) and PGF2 alpha from the time of grafting, which was accompanied by a rise in the levels of prostacyclin (PGI2, measured as its stable hydrolysis product 6-oxo-PGF1 alpha) and thromboxane A2 (TxA2, measured as its stable hydrolysis product TxB2). When the level of each individual COP in rejecting graft tissue was expressed as a percentage of the total COP measured, PGI2 and TxA2 increased from 9% to 36% and from 7% to 25%, respectively, from day three after grafting until the time of graft rejection. In contrast to these changes in COP levels in rejecting grafts, the levels of all four COP in nonrejecting allografts maintained by treatment of the graft recipients with cyclosporine remained normal--i.e., comparable to the levels in the recipients' own hearts.

Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation. However, co-administration of these two peptides to a single sponge together caused a significant increase in the rate of 133Xe clearance and angiogenesis similar to that seen with the high dose of VEGF165 (250 ng) acting alone. This VEGF/bFGF neovascular response was also blocked by daily co-administration of lavendustin A (10 jig),suramin (3 mg) or a monoclonal anti-bFGF antibody (DG2, I jig), but not lavendustin B (10 g).6 These results suggest that selective inhibition of PTK could have therapeutic potential in angiogenic diseases where VEGF plays a dominant role. Furthermore, blockade of the angiogenic activity of VEGF and VEGF,/bFGF by suramin reveals an alternative strategy in angio suppression.

[Leu8]des-Arg9-bradykinin inhibits the angiogenic effect of bradykinin and interleukin-1 in rats.

1. Subcutaneous implantation of sterile polyether sponges in rats elicited a reproducible neovascular response over 14 days, as determined by measurements of relative sponge blood flow by a 133Xe clearance technique. The angiogenic response was verified by quantitation of haemoglobin contents and histological evaluation of vascularized sponges. 2. Daily administration of 1 nmol of bradykinin (BK) into the implants significantly enhanced the basal sponge-induced neovascularization, leading to higher 133Xe clearance values, increased haemoglobin contents, cellularity and vascularity. 3. When given alone, lower doses of BK (10 pmol) or recombinant human interleukin-1 alpha (IL-1 alpha, 0.3 pmol) produced no apparent effects on the basal sponge-induced angiogenesis. However, co-administration of these two peptides produced an angiogenic response similar to that elicited by 1 nmol of BK. 4. The BK/IL-1 alpha-induced neovascularization was abolished by the bradykinin B1 receptor antagonist, [Leu8]des-Arg9-BK (1 nmol day-1), but not by the B2 receptor antagonist Ac-D-Arg-[Hyp3, D-Phe7, Leu8]-BK (1 nmol day-1). 5. Thus, if such interaction between BK and IL-1 alpha contributes to the excessive neovascularization in chronic inflammatory diseases, the blockade of B1 receptors may provide an effective treatment.

Blood flow, histamine content and histidine decarboxylase activity in rat skin grafts and their modification by cyclosporin-A.

1 An attempt has been made to monitor simultaneously the changes in blood flow, histamine content, histidine decarboxylase (HDC) activity and mast cell population in rat skin isografts and allografts. 2 The histamine content in rat skin allografts during rejection was decreased in contrast with our earlier observation in rabbits. 3 Although no definite correlation has been found between blood flow changes, histamine content or HDC activity, a reciprocal relationship appeared to exist between histamine content and HDC activity in the skin grafts, which might be a reflection of the immunosuppressive activity of this amine. 4 The immunosuppressive agent, cyclosporin-A (20 mg/kg daily) was able to prolong skin allograft survival and prevent the changes in histamine and HDC activity in allografts. 5 Possible implications of these findings are discussed in relation to current knowledge concerning the interactions between endothelial cells, lymphocytes and mast cells/basophils in graft rejection.

Effect of cyclosporin A and inhibitors of arachidonic acid metabolism on blood flow and cyclo-oxygenase products in rat skin allografts.

Using skin blood flow as a measurement of skin graft rejection in rats, it has been shown that in both isografts and allografts the blood flow at first increases above the normal, after which the flow in isografts returns to normal while that in allografts ceases at the onset of rejection. Cyclosporin A (CSA) 5-40 mg kg-1 intramuscularly produced a dose-related inhibition of graft rejection and the pattern of blood flow in the treated allografts became similar to that in isografts in that it remained about 20% above normal throughout the period of treatment. Indomethacin (Indo), inhibitor of cyclo-oxygenase and benoxaprofen (Ben), inhibitor of cyclo-oxygenase and lipoxygenase, caused an enhancement of the onset of rejection and an early cessation of blood flow in allografts. The total content of 4 cyclo-oxygenase products (COP), (prostaglandin E2 (PGE2), PGF2 alpha, 6-oxo-PGF1 alpha and thromboxane B2 (TxB2] increased both in isografts and allografts, but when individual COP were expressed as a percentage of the total, only 6-oxo-PGF1 alpha (the stable metabolite of prostacyclin) increased in allografts. This increased proportion was reduced to normal by a dose of CSA which prolonged graft survival. Indo and Ben partially inhibited COP formation and in particular that of 6-oxo-PGF1 alpha. In addition, CSA caused a dose-related inhibition of the prostacyclin produced by zymosan-activated macrophages. These findings in the rat suggest that prostacyclin is partly responsible for the increase in blood flow in allografts prior to rejection; that CSA inhibits both the recruitment of prostacyclin-producing macrophages and prostacyclin formation directly; and that inhibitors of cyclo-oxygenase enhance skin graft rejection by abrogating the immunoregulatory activity of prostacyclin.

Somatostatin receptor-mediated arachidonic acid mobilization: evidence for partial agonism of synthetic peptides.

1. Somatostatin and the stable octapeptide analogues, octreotide and angiopeptin, were examined for their ability to stimulate the release of tritium from [(3)H]-arachidonic acid pre-loaded CHO-K1 cells expressing human recombinant sst(2), sst(3) or sst(5) receptors. 2. Somatostatin stimulated tritium release (pEC(50)) through the sst(2) (7.8+/-0.1) and sst(5) (7.3+/-0.2), but not the sst(3) receptor. Octreotide behaved as a full (sst(2) receptor) or partial agonist (sst(5) receptor), whereas angiopeptin behaved as a weak partial agonist at both receptor types. 3. Maximum responses to somatostatin through both receptor types were significantly reduced by pertussis toxin, whereas pEC(50) estimates were unaffected. 4. Inhibition of MEK1 or Src, but not PKA, PI 3-kinases or tyrosine kinases, by reportedly selective inhibitors reduced sst(2)-mediated responses by somatostatin, but not angiopeptin. A selective inhibitor of PKC (Ro-31-8220) reduced both somatostatin and angiopeptin responses. 5. These data provide further evidence for partial agonist activity of synthetic peptides of somatostatin. Furthermore, the somatostatin receptor signalling mechanisms which mediate arachidonic acid mobilization appear to be multiple and complex.

Comparative studies of the angiogenic activity of vasoactive intestinal peptide, endothelins-1 and -3 and angiotensin II in a rat sponge model.

1 The angiogenic activity of four vasoactive peptides with a range of vasodilator and vasoconstrictor properties, i.e. vasoactive intestinal peptide (VIP), endothelin-1, endothelin-3 and angiotensin II, were investigated in a rat sponge model. Neovascularization was assessed by the 133Xe clearance technique and confirmed by histological studies. 2 Daily doses of the vasodilator peptide, VIP (1000 pmol), caused intense neovascularization, but a lower dose (10 pmol) produced no apparent effect. However, the lower dose of VIP, when given with a subthreshold dose of interleukin-1 alpha (0.3 pmol), produced an angiogenic response similar to that seen with the higher dose of VIP. The neovascular response induced by co-administration of VIP and interleukin-1 alpha was inhibited by simultaneous administration of 100 pmol VIP (10-28), a specific VIP receptor antagonist. 3 In contrast, daily doses of 10, 100 or 1000 pmol endothelin-3 (a mixed vasoconstrictor and vasodilator with more marked vasodilator activity) or of 100 or 1000 pmol endothelin-1 (also with mixed activity but with much more pronounced vasoconstrictor response) produced no apparent effect on sponge-induced angiogenesis. 4 The vasoconstrictor peptide, angiotensin II, in daily doses of 1000 pmol, caused an intense neovascularization like VIP but lower doses of angiotensin II (10 or 100 pmol) produced no apparent effect. The lowest dose of angiotensin II (10 pmol) when administered with the subthreshold dose of interleukin-1 alpha (0.3 pmol) had no effect on the basal neovascular response in the sponges. The angiotensin II-induced neovascular response was inhibited by co-administration of 100 nmol of the specific AT1 receptor antagonist, losartan, but not by the AT2 receptor antagonist, PD 123319. 5 These data show that VIP and angiotensin II possess angiogenic activity. However, endothelin-1 and endothelin-3 had no activity at the doses used. Thus the angiogenic response is not related to local vasoconstriction or vasodilatation in the sponges. The blockade of VIP- and angiotensin II-induced angiogenesis at the receptor level suggests that receptor modulation could provide a strategy for the management of angiogenic diseases.

Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation.

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst2(b) receptor (CHOsst2(b)) and compared these to their effects in cells expressing the sst2(a) receptor (CHOsst2(a)). In contrast to the sst2(a) receptor, the sst2(b) receptor did not mediate inhibition of bFGF (10 ng ml(-1))-stimulated re-growth and cell proliferation. Rather, SRIF (0.1-1000 nM) and angiopeptin (0.1-1000 nM) stimulated basal re-growth and proliferation of CHOsst2(b) cells in a concentration-dependent manner (estimated pEC50 values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst2 receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative effect via the sst2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst2(a) and sst2(b) receptor splice variants mediate opposite effects on cell proliferation.

Stimulation of angiogenesis by substance P and interleukin-1 in the rat and its inhibition by NK1 or interleukin-1 receptor antagonists.

1. Daily administration of 1 nmol substance P or 3 pmol recombinant human interleukin-1 alpha (IL-1 alpha) caused intense neovascularization in a rat sponge model of angiogenesis. Lower doses of substance P (10 pmol) or IL-1 alpha (0.3 pmol) were ineffective when given alone. When combined at these low doses, substances P and IL-1 alpha interacted to produce an enhanced neovascular response. 2. By use of selective tachykinin NK1, NK2 and NK3 receptor agonists, ([Sar9,Met(O2)11]substance P, [beta-Ala8]neurokinin A(4-10), Succ-[Asp6,MePhe8]substance P(6-11) (senktide), respectively), it was established that the activation of NK1 receptors is most likely to mediate the angiogenic response to substance P in this model. 3. The angiogenic activity of substance P and IL-1 alpha (10 pmol and 0.3 pmol day-1, respectively) was abolished by co-administration of (i) the selective peptide NK1 receptor antagonist, L-668,169 (1 nmol day-1), (ii) the selective non-peptide NK1 receptor antagonists, RP 67580 and (+/-)-CP-96,345 (both at 1 nmol day-1) or (iii) the IL-1 receptor antagonist, IL-1ra, (50 micrograms day-1). In contrast, the selective NK2 receptor antagonist, L-659,874 (1 nmol day-1) was ineffective. 4. The angiogenic action of substance P and IL-1 alpha was resistant to modification by mepyramine (1 nmol day-1) and/or cimetidine (10 nmol day-1), indomethacin (7 nmol day-1) or the platelet-activating factor (PAF) antagonist, WEB-2086 (22 nmol day-1), indicating that histamine, prostaglandins and PAF are not likely to be involved in this neovascular response. 5. The inhibition of the substance P/IL-1 angiogenic response by selective NK1 receptor antagonists or by an IL-1 receptor antagonist demonstrates that angiosuppression can be achieved by blocking the activity of angiogenic factors at the receptor level.

Evidence that [3H]-alpha,beta-methylene ATP may label an endothelial-derived cell line 5'-nucleotidase with high affinity.

1. In membranes prepared from a permanent cell line of endothelial origin (WEC cells), [3H]-alpha, beta-methylene ATP ([3H]-alpha, beta-meATP) labelled high (pKd = 9.5; Bmax = 3.75 pmol mg-1 protein) and low (pKd = 7.2; Bmax = 23.3 pmol mg-1 protein) affinity binding sites. The high affinity [3H]-alpha, beta-meATP binding sites in the WEC cell membranes could be selectively labelled with a low concentration of the radioligand (1 nM). In competition studies performed at a radioligand concentration of 1 nM, 88.6% of the sites possessed high affinity (pIC50 = 8.26) for alpha, beta-meATP. 2. The high affinity [3H]-alpha, beta-meATP binding sites appeared heterogeneous since in competition studies a number of nucleotide analogues (alpha, beta-meADP, ATP, ADP, AMP, GTP, GppNHp, GMP) and adenosine identified two populations of the sites labelled by 1 nM [3H]-alpha, beta-meATP. The proportion of sites with high affinity for these compounds was found to vary between 42 and 69%. 3. Approximately 60-69% of the binding sites labelled with 1 nM [3H]-alpha, beta-meATP possessed high affinity for alpha, beta-meADP (pIC50 = 8.87), AMP (pIC50 = 7.12), GMP (pIC50 = 7.34), UTP (pIC50 = 6.12), GTP (pIC50 = 7.59), GppNHp (pIC50 = 7.35) and adenosine (pIC50 = 5.45). The sites at which these compounds possessed high affinity were probably the same, since, in the presence of GMP at a concentration (10 microM) sufficient to inhibit selectively the binding of [3H]-alpha,beta-meATP, the [3H]-alpha,beta-meATP binding sites with high affinity for AMP, UTP, alpha, beta-meADP, GTP, GppNHp and adenosine were also occluded.4. WEC cell membranes were able to metabolize a trace concentration (6 nM) of [3H]-AMP to [3H]-adenosine under the conditions of the binding assay. The pIC50 values of adenosine (5.99), GMP (7.55)and the substrate AMP (7.19) for inhibiting this [3H]-AMPase activity were almost identical to their high affinity pIC50 estimates obtained in the binding assay. Although alpha, beta-meADP, alpha, beta-meATP, beta,upsilon-meATP,ATP, ADP and GppNHp identified heterogeneity in the [3H]-AMPase activity of the WEC cells, theirpIC50 values for inhibiting the major portion of the [3H]-AMPase activity were similar to their respective high affinity pIC50 values in the binding assay. It thus seems likely that WEC cells express a form of 5'-nucleotidase that possesses high affinity for both alpha,beta-meADP and alpha,beta-meATP and that this enzyme can be labelled by [3H]-alpha,beta-meATP.5. In the presence of 10 microM GMP, the affinity estimates for alpha,beta-meADP, AMP, GMP, GTP, GppNHp,ADP and adenosine at the high affinity [3H]-alpha,beta4-meATP binding sites that remained available, were lowa nd similar to their affinity estimates at the high affinity [3H]-alpha,beta-meATP binding sites of rat vas deferens. Since the high affinity [3H]-alpha,beta-meATP binding sites in rat vas deferens are thought to be P2x purinoceptors it is possible that the high affinity [3H]-alpha,beta-meATP binding sites in the WEC which possess low affinity for alpha,beta-meADP are also P2x purinoceptors.

Differential effects of angiostatic steroids and dexamethasone on angiogenesis and cytokine levels in rat sponge implants.

1. Subcutaneous implantation of sterile polyether sponges elicited a reproducible neovascular response in rats, as determined by blood flow measurement with a 133Xe clearance technique and confirmed histologically. This model was used to monitor the levels of two cytokines during angiogenesis and to compare the activities of angiostatic steroids and anti-inflammatory steroids. 2. Initial experiments followed the neovascular development over a 20-day period. Daily local injection of hydrocortisone caused a dose-dependent (0.5, 5 and 50 micrograms per sponge) inhibition of the basal sponge-induced angiogenesis. However, daily systemic treatment of hydrocortisone (2, 10 and 50 mg kg-1, s.c.) was less effective at inhibiting angiogenesis, and this inhibition was not sustained by day 20 after sponge implantation. 3. To investigate the involvement of cytokines during the course of angiogenesis, we measured the endogenous levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) in sponge implants. Levels of IL-6 and TNF-alpha peaked at day 7 and day 11 after implantation, respectively. These cytokine levels subsided through the completion of angiogenesis by day 20. 4. Subsequent experiments were carried out over a 14-day period. Among the three angiostatic steroids tested, U-24067 (6 alpha-fluoro-17,21 - dihydroxy-16 alpha-methylpregna -4,9(11)-diene-3,20-dione-21-acetate) showed a dose-dependent inhibition (0.5, 5 and 50 micrograms per sponge per day) of sponge-induced angiogenesis. Tetrahydro-S was also effective at 5 micrograms doses, but medroxyprogesterone failed to affect the angiogenic response. None of these steroids caused atrophies of the spleen and thymus. 5. Daily local injection of dexamethasone (0.5 microgram per sponge) inhibited the basal sponge-induced angiogenesis almost completely. Although higher doses of dexamethasone (5 and 50 micrograms per sponge) did not produce further inhibition of angiogenesis, they caused severe spleen and thymus weight losses, indicative of immunosuppression. 6. At the daily dose of 5 micrograms per sponge, dexamethasone inhibited angiogenesis and produced a marked reduction in the levels of TNF-alpha and IL-6 at day 14. In contrast, hydrocortisone, U-24067 and tetrahydro-S did not influence the levels of TNF-alpha and IL-6. 7. We concluded that the anti-angiogenic activity of angiostatic steroids and anti-inflammatory steroids in the rat sponge model is independent of their ability to reduce the production of TNF-alpha and IL-6. The differential effects of angiostatic and anti-inflammatory steroids suggest that U-24067 and its derivatives may have therapeutic potential in the management of angiogenic diseases such as rheumatoid arthritis.

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells.

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst5 receptors (CHOsst5), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2. In VSMC, SRIF (0.1 nM - 1 microM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC50 8.0-8.6) of the stimulated regeneration induced by submaximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml[-1]), platelet-derived growth factor-BB (PDGF, 5 ng ml[-1]) or endothelin-1 (ET-1, 100 nM). SRIF (pIC50 8.8) also inhibited bFGF-induced regeneration of CHOsst5 cells. 3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml[-1]) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM - 1 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg ml[-1]). 4. The sst5 receptor-selective agonist, L-362,855 (pIC50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst5 cells whilst the sst2 receptor-selective agonist, BIM-23027 (pIC50 6.8), was approximately 20 times weaker than SRIF. 5. The sst5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst5 cells (estimated pKB values 8.8 and 8.3, respectively). 6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml[-1]) for 20 h. 7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst5 receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive Gi/Go proteins.