The Effect Of Choline On The Angiogenesis Induced By Cerebral Ischemia And Pharmacological Mechanism Investigation

Background:Stroke is characterized as high incidence, high mortality and high disability. Ischemic stroke is the overwhelming type of stroke, accounting for around 87% of all strokes. Stroke is the second leading death cause in the population older than 60 years and the fifth leading cause of death in those people aged 15 to 59 years old. In China, stroke has become the third cause of death in the people of cities and the second one in rural areas.At present, in our country there are 7.5 million stroke patients, of which 4.5 million patients lose the ability of work or self-care. Each year new cases of stroke are about 2.5 million. The incidence rate of ischemic stroke is increasing by 8.7% annually on average. Human health was impacted severely by stroke, which has been the significant burden for patients and their families and country in medical treatment, economy and society. Now the annual cost of stroke care in China is approximately 40 billion RMB, ten times higher than the care of other cardiovascular diseases.The pathogenesis of ischemic stroke is extremely complicated and closely related with energy exhaustion, oxidative stress, toxic effects of excitatory amino acids, nitric oxide, calcium overload, apoptosis and inflammation and so on. Thrombolysis is an effective treatment for ischemic stroke in clinic. At present, the only thrombolytic drug FDA-approved is tissue plasminogen activator(t PA). Because the therapeutic time window of t PA is within 4.5 h, the majority of patients did not receive effective treatment timely. Although in the last several decades substantial efforts have been invested in developing neuroprotective drugs, these efforts have not resulted in clinically-efficacious therapies for ischemic stroke. These failures highlight the need for development of new therapeutic strategy and medicine for ischemic stroke, which has become the focus and difficult areas.Although ischemia and hypoxia can induce compensatory angiogenesis in the brain when ischemic stroke occurs, the process is very slow and weak. Using drugs to induce angiogenesis in ischemic brain may create new collateral circulation result in relieving nerve damage caused by ischemia and producing a therapeutic effect on nerve regeneration and recovery of neurological function, which was called therapeutic angiogenesis. Therapeutic angiogenesis is promising to become a new approach of clinical treatment for ischemic stroke. Previous study inour lab reported that α7 n ACh R existing in endothelial cells participated in angiogenesis regulation,its agonist choline could increase intracellular calcium concentration in endothelial cells, and promote cell proliferation, tube formation in vitro and new collateral circulation created in myocardial infarction tissue. These results suggested that α7 n ACh R might become a new target of treatment for ischemic cardiovascular and cerebrovascular diseases. It is worth further study that whether choline can treat ischemic stroke through the mechanism of promoting angiogenesis in ischemic brain. In this study, rats with permanent middle cerebral artery occlusion and brain microvascular endothelial cells of rats cultured under hypoxia were introduced to observe the effects of choline on the treatment and angiogenesis-related mechanism for ischemic stroke.Methods: 1. Animal experimental protocolMale Sprague–Dawley rats, weighing 280±20g were randomly divided into eight groups: sham-operated group, rats underwent a similar surgery without p MCAO and treated with vehicle(sham); model group, rats were received by p MCAO and treated with vehicle(model); p MCAO rats treated with 50, 100, and 200 mg/kg/d choline(Cho50, Cho100 and Cho200, respectively); p MCAO rats treated with 20 mg/kg/d nimodipine(Nim); p MCAO rats treated with 100 mg/kg/d choline plus 1 mg/kg/d MLA,a selective α7 n ACh R antagonist(Cho100+MLA); p MCAO rats treated with 1 mg/kg/d MLA(MLA). The model of p MCAO was received by right middle cerebral artery occlusion with a filament technique. Drugs were dissolved in sterile water for injection. Choline or nimodipine were administered orally, while MLA was administered subcutaneously. The rats were treated with drugs or vehicle 4 h after surgery, then once a day in 2 m L/kg for 10 days.2. Detection index in animal experimentsA total of 144 rats were used for the survival rate study and determining the body weight. This measurement was terminated 10 days after p MCAO. Rats were evaluated for neurological deficits daily with Bederson’s score after p MCAO. Behavioral assessment consisted of bilateral grasp and beam walking test. The brains were quickly removed when the rats were euthanized and sacrificed 10 days after p MCAO. The rat brains were sectioned into coronal sections and stained using TTC saline solution to measure the infarct volume rates. The photos of ischemic hemispheres surface were taken with a digital camera and were calculated for the vascular area, total blood length using Image-Pro plus 6.0 software for analyzing the blood vessel density. The brain sections were stained by CD34 immunohistochemistry and CD34/PCNA double labeling immunofluorescence to investigate the capillary density. NO and VEGF levels in rat serum were assayed by NO biochemistry or VEGF ELISA kit. The m RNA and protein expression of α7 n ACh R,,HIF-1α,e NOS,i NOS and VEGF in the ischemic brain cortex of p MCAO rats were measured by real-time PCR and Western blot.3. Culture and identification of primary r BMECsPrimary rat brain microvascular endothelial cells(r BMECs) were isolated from rat brains of 3 weeks old SD rats by using a modified collagenase/dispase-based digestion protocol. Briefly, cerebral cortex from 3 weeks old SD rat was minced, homogenized and passed through 200 μm and 77 μm strainers. The tissue left on the strainer was digested with 0.1% collagenase/dispase solution for 25 min at 37 °C. The pellet was separated by centrifugation(1000×g, 20 min) in 25% bovine serum albumin-Dulbecco’s modified Eagle medium. The precipitation was cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2 with complete DMEM culture medium(20% fetal bovine serum, 100 μg/m L endothelial cell growth factor, 100 μg/m L heparin, 3.75 mg/m L hydroxyethyl piperazine ethanesulfonic acid, 0.2 U/m L insulin, 0.3 mg/m L L-glutamine, 100 U/m L penicillin and 100 μg/m L streptomycin, p H 7.2–7.4). Cells used in the present study were passaged three times and were positive with immunocytochemistry for CD34.4. Detection index in cell experimentsUnder the condition of hypoxia(1% O2, 95% CO2), using small-interfering-RNA(si RNA) targeting HIF-1α, the effect of choline on the proliferation of r BMECs was detected using MTS kit, the effect of choline on the migration of r BMECs was measured by wound healing assay. We applied Matrigel Matrix Growth Factor Reduced to induced tube formation of r BMECs and investigated the effect of choline on the tube formation. NO and VEGF levels were detected by NO biochemistry kit and VEGF ELISA kit. Cells were treated with the following conditions: Control(DMEM without choline); 1 μM choline; 10 μM choline; 100 μM choline; 10 μM choline plus 1 μM MLA; 1 μM MLA; 10 μM choline plus 100 nmol/L HIF-1α si RNA.5. Statistical analysisAll data were presented as mean±SD. Statistical analysis of the data with SPSS13.0 software package was performed using one-way ANOVA analysis for differences among groups and Bonferroni’s test for comparing between two groups. Differences were considered significant at P<0.05.ResultsResults:Part ⅠThe effects of choline on the treatment and related-angiogenesis mechanism forp MCAO rats1. Choline increased survival rate,improved body weight and neurological functionThe survival of rates was reduced to 60% 10 days after p MCAO compared with sham group. Choline(50～200 mg/kg) administrated orally for 10 days improved the survival rate to 67%～80%, however MLA at the dose of 1 mg/kg daily could prevent the effect of 100 mg/kg choline. The body weights of the rats decreased significantly at 10 days after p MCAO compared with the rats in the sham group. Treatment with 100-200 mg/kg/d choline for 10 days led to marked elevations in body weight. The neurological deficit was evaluated with Bederson’s scores for 10 days. Bederson’s scores increased siglificantly in the p MCAO rats. The neurological deficit was prevented by treated daily with choline at the dose of 100 or 200 mg/kg for 7～10 days. Bilateral grasp and beam walking test were applied to assess the forelimb strength and sensorimotor reflexes, coordination respectively. The muscle strength scores were markedly elevated by choline in dose dependent manner compared with p MCAO rats after 10 days. These features of choline on the neurological function were antagonized completely by MLA. However choline did not change the scores in the beam walking test significantly.2. Choline decreased infarct volume and neurons deathIn the p MCAO group rats, the infarct volumes were significantly increased compared with sham-operated rats. Choline could notably decrease the infarct volume dose-dependently in rats 10 days after p MCAO, but which was reversed by treating with MLA. In the microscopic images with HE staining, there are many died neurons accompanied with morphological changes of nuclear shrinkage, swelling and vacuolization in the p MCAO rats. These changes were prevented by choline dramatically; however MLA could block these effects.3. Choline accelerate angiogenesis in ischemic brainThe effect of choline on the angiogenesis on the surface and in penumbra of rat ischemic brain cortex was detected 10 days after p MCAO. More vessel branchs were observed in the p MCAO group than that in the sham group. Choline could develop this change in dose-dependently. Compared with p MCAO, the total vessel areas and lengths were significantly increased by choline. Microvessel density was determined by the immunofluorescence of CD34/PCNA double labeling, which showed the new proliferative vascular endothelial cells. The number of co-localization cells increased notably in the choline group at the dose of 100 and 200 mg/kg compared with p MCAO group. The effect of choline promoting angiogenesis in ischemic brain of p MCAO rats was prevented by MLA. The result of CD34 single labeled immunohistochemical staining was consistent with the above. Nimodipine did not have the same role as choline.4. Choline increase NO and VEGF levels in serumNO and VEGF levels in serum of rats were determined 10 days after p MCAO. Compared with sham group, NO level decreased obviously, but VEGF did not change significantly. Choline at 100～200 mg/kg could elevate NO and VEGF levels notably in rats’ serum 10 days after p MCAO. These effects of choline were blocked by MLA. Nimodipine increased the level of NO but did not change that of VEGF.5. Effects of choline on gene and protein expression in ischemic brain cortexThe ischemic brain cortex of rats were extracted and examinated for relative gene and protein expression 10 days after p MCAO. The m RNAs of α7 n ACh R, HIF-1α, and VEGF were apparently upregulated after p MCAO compared with the sham group. However, e NOS m RNA level was downregulated notebaly and i NOS gene did not change obviously in the p MCAO rats. Choline at 100 mg/kg increased significantly the m RNA expression of α7 n ACh R, HIF-1α, e NOS and VEGF, but had no effect on i NOS gene. The upregulation effects of choline on gene expression were abolished by treatment with 1 mg/kg MLA, but which had no effects on these gene expressions under the same conditions. The results of protein expression of α7 n ACh R, HIF-1α, e NOS, i NOS and VEGF in all groups were consistent in principle with those of gene expression.Part ⅡThe effects of choline on rBMECs under hypoxia condition1. Choline promote the proliferation, migration and tube formation of r BMECsUnder the hypoxia condition with the oxygen concentration of 1%, the proliferation, migration and tube formation of r BMECs were facilitated significantly by the treatment of choline(1～100 μM) in a concentration-dependent manner, which could be prevented by MLA at 1 μM or by 100 nmol/L HIF-1α si RNA.The MLA or HIF-1α si RNA at such dose had no direct effects for r BMECs. The results suggested that hypoxia-induced r BMECs proliferation, migration and tube formation might be promoted by choline upregulating HIF-1α expression through activating α7 n ACh R.2. Choline increase NO and VEGF in surpernatant of r BMECsUnder the hypoxia condition with the oxygen concentration of 1%, r BMECs were incubated with choline by the present of MLA or HIF-1α si RNA. NO and VEGF in surpernatants of r BMECs were measured after 24 h. Compared with control group, choline(1～100 μM) significantly elevated the levels of NO and VEGF in a concentration-dependent manner. MLA at 1 μM could prevent the effects of choline on the release of NO and VEGF, but HIF-1α si RNA at 100 nmol/L only prevented the release of VEGF, which suggested that the effect of choline promoting the release of VEGF was related with activating α7 n ACh R and upregulating HIF-1α, but the effect of choline promoting the release of NO was only related with activating α7 n ACh R.The effects of choline on the release of NO and VEGF were abolished by MLA 1 μM, which had no direct effects on the release of NO and VEGF. It suggested that these effects of choline were relative with α7 n ACh R activated.Conclusions:1. Choline could promote angiogenesis and collateral circulation establishment in ischemic brain of p MCAO rats, which increased survival rates of rats, decreased brain infarct volumes and improved body weights, neurological and behavioral functions.2. The angiogenesis-related mechanisms of choline on treatment for ischemic stroke were related with upregulations of m RNA and protein expression in ischemic brain and VEGF, NO levels in serum of p MCAO rats.3. Choline could promote r BMECs proliferation, migration, tube formation and releases of NO and VEGF via activating α7 n ACh R, which might become an important cellular mechanism for cerebral ischemic angiogenesis.4. Enhancing angiogenesis in ischemic brain by activating α7 nAChR pathway might become a new approach to treat ischemic stroke.