Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuetong (SXT) injection is formulated by leech and earthworm, has been widely used in the treatment of thrombotic cardiovascular and cerebrovascular diseases with remarkable clinical efficacy. AIM OF THE STUDY: The purpose of this study is to investigate the protective mechanism of SXT injection on the mice model of hindlimb ischemia, and to evaluate the angiogenic effects of SXT injection and its main active substances. MATERIALS AND METHODS: Hindlimb ischemia was induced by left femoral artery ligation. After operation, the mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system. Tissue recovery and capillary density were evaluated by histological and immunofluorescent staining. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF-BB) expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Human umbilical vein endothelial cells (HUVECs) proliferation was measured using a BrdU kit and the viability of HUVECs was performed by MTT assay. Migration of HUVECs was performed by the wound healing method and a modified transwell assay. Capillary tube formation by HUVECs was examined by using Matrigel assay. Western blotting was used to detect the expressions of p-Cofilin, p-MYPT1, and p-LIMK1. RESULTS: SXT injection treatment significantly restored the blood flow and reduced tissue injury in mouse gastrocnemius muscle. SXT injection treatment increased capillary density and promoted angiogenesis in hindlimb ischemia. Moreover, SXT injection enhanced the expression of VEGF-A and PDGF-BB at both mRNA and protein levels in ischemic tissue of mice. SXT injection and its main active peptides dramatically increased the migration and capillary tube formation of HUVECs. SXT injection and its peptides enhanced protein expressions of the phosphorylation of MYPT1, Cofilin, and LIMK1. DSYVGDEAQSKR, YNELRVAPEEHP, and IQFLPEGSPVTM may act as the active components of SXT injection. CONCLUSION: SXT injection promoted angiogenesis and improved function recovery in hindlimb ischemia mice by regulation of VEGF-A/PDGF-BB. Moreover, SXT injection and its active peptides induced cell migration and tube formation in HUVECs through activating the MYPT1/LIMK1/Cofilin pathway. This study provided experimental basis for SXT injection in the treatment of ischemic diseases and revealed the effective substance of SXT injection in regulating angiogenesis, providing better evidence for the clinical application of SXT injection.

Cadmium-induced hypertension is associated with renal myosin light chain phosphatase inhibition via increased T697 phosphorylation and p44 mitogen-activated protein kinase levels.

Dietary intake of the heavy metal cadmium (Cd2+) is implicated in hypertension, but potassium supplementation reportedly mitigates hypertension. This study aims to elucidate the hypertensive mechanism of Cd2+. Vascular reactivity and protein expression were assessed in Cd2+-exposed rats for 8 weeks to determine the calcium-handling effect of Cd2+ and the possible signaling pathways and mechanisms involved. Cd2+ induced hypertension in vivo by significantly (p < 0.001) elevating systolic blood pressure (160 ± 2 and 155 ± 1 vs 120 ± 1 mm Hg), diastolic blood pressure (119 ± 2 and 110 ± 1 vs 81 ± 1 mm Hg), and mean arterial pressure (133 ± 2 and 125 ± 1 vs 94 ± 1 mm Hg) (SBP, DBP, and MAP, respectively), while potassium supplementation protected against elevation of these parameters. The mechanism involved augmentation of the phosphorylation of renal myosin light chain phosphatase targeting subunit 1 (MYPT1) at threonine 697 (T697) (2.58 ± 0.36 vs 1 ± 0) and the expression of p44 mitogen-activated protein kinase (MAPK) (1.78 ± 0.20 vs 1 ± 0). While acetylcholine (ACh)-induced relaxation was unaffected, 5 mg/kg b.w. Cd2+ significantly (p < 0.001) attenuated phenylephrine (Phe)-induced contraction of the aorta, and 2.5 mg/kg b.w. Cd2+ significantly (p < 0.05) augmented sodium nitroprusside (SNP)-induced relaxation of the aorta. These results support the vital role of the kidney in regulating blood pressure changes after Cd2+ exposure, which may be a key drug target for hypertension management. Given the differential response to Cd2+, it is apparent that its hypertensive effects could be mediated by myosin light chain phosphatase (MLCP) inhibition via phosphorylation of renal MYPT1-T697 and p44 MAPK. Further investigation of small arteries and the Rho-kinase/MYPT1 interaction is recommended.

Folium Sennae and emodin reverse airway smooth muscle contraction.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide-independent pathways.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is one of the main compounds present in Artemisia species. Eupatilin has both antioxidative and anti-inflammatory properties and a relaxation effect on vascular contraction regardless of endothelial function. We evaluated the relaxant effects of eupatilin on the corpus cavernosum (CC) of rabbits and the underlying mechanisms of its activity in human corpus cavernosum smooth muscle (CCSM) cells. Isolated rabbit CC strips were mounted in an organ bath system. A conventional whole-cell patch clamp technique was used to measure activation of calcium-sensitive K+ -channel currents in human CCSM cells. The relaxation effect of eupatilin was evaluated by cumulative addition (10-5  m ~ 3 × 10-4  m) to CC strips precontracted with 10-5  m phenylephrine. Western blotting analysis was performed to measure myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI-17) expression and to evaluate the effect of eupatilin on the RhoA/Rho-kinase pathway. Eupatilin effectively relaxed the phenylephrine-induced tone in the rabbit CC strips in a concentration-dependent manner with an estimated EC50 value of 1.2 ± 1.6 × 10-4  m (n = 8, p < 0.05). Iberiotoxin and tetraethylammonium significantly reduced the relaxation effect (n = 8, p < 0.001 and p = 0.003, respectively). Removal of the endothelium or the presence of L-NAME or indomethacin did not affect the relaxation effect of eupatilin. In CCSM cells, the extracellular application of eupatilin 10-4  m significantly increased the outward currents, and the eupatilin-stimulated currents were significantly attenuated by treatment with 10-7  m iberiotoxin (n = 13, p < 0.05). Eupatilin reduced the phosphorylation level of MYPT1 at Thr853 of MLCP and CPI-17 at Thr38. Eupatilin-induced relaxation of the CCSM cells via NO-independent pathways. The relaxation effects of eupatilin on CCSM cells were partially due to activation of BKCa channels and inhibition of RhoA/Rho-kinase.

Baicalein potently inhibits Rho kinase activity and suppresses actin stress fiber formation in angiotensin II-stimulated H9c2 cells.

Baicalein is a flavonoid (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran4-one) and an active principle in Scutellaria baicalensis. The present study was performed to investigate the mechanisms underlying the anti-fibrotic effects of baicalein with a focus on Rho kinase (ROCK) inhibition. The effect of baicalein on ROCK activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The underlying mechanisms of baicalein were examined using angiotensin II-stimulated H9c2 cells. Rho kinase (ROCK1 and ROCK2) studies using IMAP-TR-FRET showed that baicalein possesses potent ROCK inhibitory activity with IC50 values of 6.55 and 2.82 µM, respectively. Pretreatment with baicalein (for 2 h) concentration-dependently decreased the angiotensin II-induced phosphorylation of myosin phosphatase (MYPT) and myosin light chain (MLC). Furthermore, baicalein also concentration-dependently suppressed actin stress fiber formation in angiotensin II-stimulated H9c2 cells. These results suggest that baicalein potently inhibits ROCK and that by so doing it modulates actin stress fiber formation. These anti-fibrotic effects of baicalein explain, at least in part, its pharmacology and mode of action.

Combining curcumin (diferuloylmethane) and heat shock protein inhibition for neurofibromatosis 2 treatment: analysis of response and resistance pathways.

Neurofibromatosis type 2 (NF2) is a genetic condition characterized by inactivation of the NF2 tumor suppressor gene and the development of schwannomas. The NF2 gene product, merlin, is activated (dephosphorylated) by contact inhibition and promotes growth suppression. We investigated the effect of curcumin (diferuloylmethane), a molecule with anti-inflammatory and antitumorigenic properties, on human schwannoma cell growth and the regulation of merlin by curcumin in both NF2 cells and neuroblastoma (non-NF2) cells. Curcumin inhibited the growth of HEI-193 schwannoma cells in vitro and downregulated the phosphorylation of Akt and extracellular signal-regulated kinase 1/2. Curcumin also activated MYPT1-pp1δ (a merlin phosphatase), which was associated with dephosphorylation of merlin on serine 518, an event that results in the folding of merlin to its active conformation. In addition, curcumin induced apoptosis and generated reactive oxygen species in HEI-193 cells. Consequently, hsp70 was upregulated at the mRNA and protein levels, possibly serving as a mechanism of escape from curcumin-induced apoptosis and growth inhibition. Endogenous merlin and hsp70 proteins interacted in HEI-193 schwannoma and SK-N-AS neuroblastoma cells. The combination of curcumin and an hsp inhibitor synergistically suppressed schwannoma cell growth. Our results provide a rationale for combining curcumin and KNK437 in the treatment of NF2.

Honokiol attenuates vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway in rat aortic rings.

OBJECTIVES: Honokiol is a small-molecule polyphenol isolated from the species Magnolia obovata. We hypothesized that honokiol attenuated vascular contractions through the inhibition of the RhoA/Rho-kinase signalling pathway. METHODS: Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. Phosphorylation of 20kDa myosin light chains (MLC(20) ), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17kDa (CPI17) were examined by immunoblot. We also measured the amount of guanosine triphosphate RhoA as a marker for RhoA activation. KEY FINDINGS: Pretreatment with honokiol dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A(2) agonist U46619. Honokiol decreased the phosphorylation levels of MLC(20) , MYPT1(Thr855) and CPI17(Thr38) as well as the activation of RhoA induced by 8.0mm NaF or 30nm U46619. CONCLUSIONS: These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway.

The mechanism of vasorelaxation induced by Schisandra chinensis extract in rat thoracic aorta.

AIM OF THE STUDY: Schisandra chinensis (SC) is a known medical herb for the treatment of cardiovascular symptoms associated with menopausal symptoms in Korea. However, the pharmacological action mechanisms involved have not been well studied. This study was aimed to investigate the vascular effects of SC in rat thoracic aorta. MATERIALS AND METHODS: We isolated the hexane, chloroform, and methanol extracts from SC and evaluated their vasodilatory effects in the rat thoracic aorta. RESULTS: Hexane extracts of SC (SCHE, 5 x 10(-5) to 10(-3) g/L) caused a concentration-dependent relaxation in both endothelium-intact and -denuded aortas. The relaxant effect of SCHE on the endothelium-intact aorta was more prominent than on the endothelium-denuded aorta. The former was significantly attenuated by L-NAME, a nitric oxide synthase inhibitor, and ODQ, a soluble guanyl cyclase inhibitor, but not by tetraethylammonium, a nonselective blocker of K(+) channels, and indomethacin, a cyclooxygenase inhibitor. Furthermore, SCHE caused nitrite production as well as eNOS activation in aortic segments, suggesting implication of NO signal pathway in SCHE-induced relaxation. In endothelium-denuded aorta, SCHE-induced vasorelaxation was also attenuated by calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, but not by ML-9, a MLC kinase inhibitor, suggestive of implication of MLC phosphatase activation. Phenylephrine-enhanced MLC phosphorylation ratio was significantly attenuated by SCHE, which was recovered to the control level by pretreatment with calyculin A. CONCLUSIONS: Taken collectively, these findings suggest that the vascular relaxation evoked by SCHE was mediated by not only endothelium dependent NO pathway but also direct effect on vascular smooth muscle cell via dephosphorylation of MLC.

[Screening, cloning and identification of the human endometrial carcinoma-related genes].

OBJECTIVE: To screen, clone and identify the cDNA fragments of human endometrial carcinoma-related genes,and explore the molecular mechanism of endometrial carcinogenesis. METHODS: Pure endometrial glandular epithelial cells and endometrial carcinoma cells were obtained by laser capture microdissection (LCM). RNA from these cells was isolated, and differentially expressed gene fragments that were specialy relevant to endometrial carcingenesis were identified by using fluorescence differential display reverse transcription polymerase chain reaction (FDD-PCR). The selected fragments were cloned, sequenced and verified by reverse Northern blot analysis, and positive fragments were BLAST analysed and compared with those in Genbank. RESULTS: 38 differential fragments were isolated, 3 of which were expressed more abundantly in normal endometrium and 35 were highly expressed in endometrial carcinoma. 10 fragments were recoverd, cloned and sequenced, confirmed by reverse Northern blot analysis, among which 6 fragments were positive. BLAST analysis showed that T1.1 was homologous to cyclin-dependent protein kinase 7 (CDK7, 99%); L1.9 was homologous to protein phosphatase 1 regulatory (inhibitor) subunit 12A (PPP1R12A, 99%); L1.21 and L1.22 were homologous to cellular repressor of E1A-stimulated genes 1 (CREG, 100%); L1.25 and L1.26 were homologous to solute carrier family 39 (zinc transporter) member 10 (SLC39A10, >98%). CONCLUSION: Gene fragments related to endometrial carcinoma have been obtained by applying LCM and FDD-PCR. To our knowledge it is the first time that the correlation between CDK7, PPP1R12A, CREG, SLC39A10 and endometrial carcinoma is discovered at mRNA level, and their role in molecular mechanism of cancinogenesis is discussed. CDK7, CREG, SLC39A10 as new candidate oncogene and PPP1R12A as new candidate anti-oncogene are worthy of being further investigated in the future.

Linalyl acetate as a major ingredient of lavender essential oil relaxes the rabbit vascular smooth muscle through dephosphorylation of myosin light chain.

In a preliminary experiment, we found that lavender essential oil relaxes vascular smooth muscle. Thus, the present experiments were designed to investigate the relaxation mechanism of linalyl acetate as the major ingredient of lavender essential oil in rabbit carotid artery specimens. Linalyl acetate produced sustained and progressive relaxation during the contraction caused by phenylephrine. The relaxation effect of linalyl acetate at a concentration near the EC50 was partially but significantly attenuated by nitroarginine as an inhibitor of nitric oxide synthase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one as an inhibitor of guanylyl cyclase, or by the denudation of endothelial cells. In specimens without endothelium, the phenylephrine-induced contraction and phosphorylation of myosin light chain (MLC) were significantly attenuated after the pretreatment with linalyl acetate. The relaxation caused by linalyl acetate in the endothelium-denuded specimens was clearly inhibited by calyculin A as an inhibitor of MLC phosphatase, although not by ML-9 as an inhibitor of MLC kinase. Furthermore, suppression of the phenylephrine-induced contraction and MLC phosphorylation with linalyl acetate was canceled by the pretreatment with calyculin A. These results suggest that linalyl acetate relaxes the vascular smooth muscle through partially activation of nitric oxide/cyclic guanosine monophosphate pathway, and partially MLC dephosphorylation via activating MLC phosphatase.

Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury.

Axonal regeneration within the injured central nervous system (CNS) is hampered by multiple inhibitory molecules in the glial scar and the surrounding disrupted myelin. Many of these inhibitors stimulate, either directly or indirectly, the Rho intracellular signaling pathway, providing a strong rationale to target it following spinal cord injuries. In this study, we infused either control (PBS) or a ROCK inhibitor, Y27632 (2 mM or 20 mM, 12 microl/day for 14 days) into the intrathecal space of adult rats starting immediately after a cervical 4/5 dorsal column transection. Histological analysis revealed that high dose-treated animals displayed significantly more axon sprouts in the grey matter distal to injury compared to low dose-treated rats. Only the high dose regimen stimulated sprouting of the dorsal ascending axons along the walls of the lesion cavity. Footprint analysis revealed that the increased base of support normalized significantly faster in control and high dose-treated animals compared to low dose animals. Forepaw rotation angle, and the number of footslips on a horizontal ladder improved significantly more by 6 weeks in high dose animals compared to the other two groups. In a food pellet reaching test, high dose animals performed significantly better than low dose animals, which failed to recover. There was no evidence of mechanical allodynia in any treatment group; however, the slightly shortened heat withdrawal times normalized only with the high dose treatment. Collectively, our data support beneficial effects of high dose Y27632 treatment but indicate that low doses might be detrimental.