血管生長包含幾個機轉例如血管新生（angiogenesis）、以及血管系形成（vasculogenesis）。血管新生主要是在既有的血管上萌發新生血管，基本上認為和組織缺血後血管新生因子的調控有關。
Cilostazol是目前治療周邊動脈狹窄引致間歇性跛行的常規用藥，主要因為具有抗血小板以及血管擴張效果。其藥理作用機轉乃是抑制第三型磷酸雙酯酶（phosphodiesterase type III），以增加細胞內環狀單磷酸腺苷（cyclic AMP）。特別的是很多證據顯示，該藥品尚有很多細胞效應。
在本論文研究， 我們的結果顯示出cilostazol有促進HUVECs細胞增生,移行,有效在體外增加血管形成並且增加cAMP和NO的表現。 我們發現cilostazol是由PIK 3/Akt將eNOS磷酸化而引發HUVECs的趨化作用。 同時我們也使用小鼠下肢缺血模式探討cilostazol的血管新生及血管系形成效應， 這些結果暗示著cilostazol是可以有效的促進下肢血管新生本研究有關。 cilostazol的探討是創新且特別的，對於學術將具重大衝擊，並且將來可能幫助cilostazol在其他重大組織缺血如心肌缺血梗塞的基礎和臨床研究，以造福廣大人類。

Blood vessel growth is mediated by angiogenesis, which is defined as the formation of new blood vessel out of existing vessels, as well as vasculogenesis, a process that circulating progenitor cells contributes to adult neovascularization.
Cilostazol, a commercially available drug holding antiplatelet and vasodilating effects, increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibiting the activity of phosphodiesterase 3. Interestingly, this chemical compound has a lot of cellular effects.
In current work, we demonstrated that cilostazol promoted proliferation and migration of human umbilical cord vein endothelial cells (HUVECs), enhanced in-vitro vascular tube formation, and increased releasing of cAMP and NO from them. Furthermore, cilostazol activated eNOS and PI3-K/Akt signaling pathways. We also examined the angiogenic and vasculogenic effects of cilostazol in a murine hindlimb ischemia model.
Our data showed that cilostazol enhanced angiogenesis and vasculogenesis with resultant flow recovery after murine hindlimb ischemia partly mediated by promoting mobilization of bone marrow-derived stem cells into circulation and increasing in situ expression of some proteins involved in angiogenesis. In addition, cilostazol significant increased colony forming unit of human endothelial progenitor cells. These results are unique and clinically significant with potential in translational therapy. According to our report, further preclinical and clinical studies of cilostazol on the other ischemic situations such as myocardial infarction will be justified.

Abstract ------------------------------------------------------------------------------------ 1
Abstract in Chinese----------------------------------------------------------------------2
Acknowledgements-----------------------------------------------------3
Abbreviation----------------------------------------------------------4
Chapter 1 Introduction
Ⅰ. Angiogenesis---------------------------------------------------------------------9
Ⅱ. The pharmacology of cilostazol ------------------------------------------------10
Ⅲ. Signal transductions involved in angiogenesis---------------------------------------12
Chapter 2 Material and Methods
I. Reagents-----------------------------------------------------------------------------------16
II. Antibody----------------------------------------------------------------------------------16
III. Assay kits--------------------------------------------------------------------------------17
IⅤ. Preparation of medium, buffer, and solution--- -----------------------------------17
V. Amimals and amimals care -----------------------------------------------------------19
Methods
I. Isolation and counting of endothelial progenitor cells -----------------------------19
II. Culture of human umbilical cord vein endothelial cells---------------------------20
III. Cell proliferation assay----------------------------------------------------------------20
IV. Chemotaxis assay----------------------------------------------------------------------20
V. Measurement of nitric oxide production---------------------------------------------21
VI. Measurement of cyclic AMP (cAMP) level ---------------------------------------21
VII. Tube formation -----------------------------------------------------------------------21
VIII. Assays of eNOS and AKT phosphorylation in HUVECs----------------------21
IX. Mouse hindlimb ischemia model----------------------------------------------------22
X. Measurement of blood flow enhancement------------------------------------------22
XI. Measurement of capillary density in the ischemic leg---------------------------22
XII. Gelatin-base zymography---------------------------------------------------------- 23
XIII. Western blot analysis of ischemic muscles -------------------------------------23
XIV. Flow cytometery analysis of detect peripheral blood CD34+ mononuclear cells------------------------------------------------------------------------------------24
Statistical Analysis----------------------------------------------------------24
Cpapter 3 Results
I. Cilostazol increased early human EPC numbers------------------------------------25
II. Effect of cilostazol on HUVEC proliferation---------------------------------------25
III. Effect of cilostazol on HUVECs migration----------------------------------------25
IV. Cilostazol induced nitric oxide production----------------------------------------26
V. Cilostazol induced cyclic-AMP production----------------------------------------26
VI. Cilostazol promoted vascular tube formation on Matrigel----------------------26
VII. Cilostazol induced phosphorylation of eNOS and Akt on HUVECs.--------27
VIII. Enhancement of blood flow recovery by cilostazol in hindlimb ischemia
ICR mice----------------------------------------------------------------------------27
IX. Cilostazol normalizede VEGF expression and eNOS phosphorylation in ischemic
muscle of ICR mice-------------------------------------------------------------------28
X. Cilostazol enhanced MMP-2 and MMP-9 activity in ischemic muscle in ICR mice-------------------------------------------------------------------------------------28
XI. Capillary density was significantly increased in cilostazol treated mice------28
XII. Cilostazol increased of CD34+ cells ----------------------------------------------29
Chapter 4 Discussion-------------------------------------------------------30
References-------------------------------------------------------------------------------35
Figures------------------------------------------------------------------------------------44

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