Peripheral Blood Autologous CD34+ Cell Transplantation Promotes Angiogenesis With Atherosclerotic Ischemia for Elderly
Peripheral Blood Autologous CD34+ Cell Transplantation Promotes Angiogenesis With Atherosclerotic Ischemia for Elderly
To verify angiogenesis in elderly patients with atherosclerotic ischemia after peripheral
 blood autologous CD34+ cell transplantation.
History and current related studies Arteriosclerosis obliterans is a kind of arterial lumen
 stenosis and occlusive disease, which is caused by the continuous expansion of arterial
 atherosclerosis and secondary thrombosis. Clinical manifestations are local skin temperature
 reduction, intermittent claudication, rest pain, ulcers difficult to heal, and gangrene
 infection.Some patients require amputation or even it is life-threatening. Arteriosclerosis
 obliterans of lower limbs are commonly treated by surgical treatment, endovascular treatment
 and non-surgical treatment, but the effect is not very satisfactory.

 For the treatment of arteriosclerosis obliterans, stem cell transplantation has achieved
 great progress in angiogenesis and collateral circulation. There are two main mechanisms of
 stem cells involved in angiogenesis: (1) Promoting differentiation and maturation of new
 blood vessels by homing and integration in the damaged vascular plexus; (2) Promoting
 angiogenesis in ischemic tissue by paracrine of vascular endothelial growth factor (VEGF).In
 recent years, because of the discovery of endothelial progenitor cells in peripheral blood,
 the concept of angiogenesis has been renewed. Transplantation of endothelial progenitor cells
 and bone marrow cells for angiogenesis has become the focus of research. Endothelial
 progenitor cell and bone marrow cell transplantation have been used to promote angiogenesis
 in ischemic tissue in order to treat limb ischemia.

 Bone marrow stem cell mobilization is used to promote the replication of bone marrow stem
 cells, so as to increase the number of endothelial progenitor cells in peripheral blood,
 i.e., the number of stem cells homing to ischemic tissue, and finally accelerating the speed
 and magnitude of angiogenesis. Fujisaki et al. have confirmed that bone marrow cell
 mobilizers can significantly stimulate bone marrow hematopoiesis and increase the number of
 peripheral blood stem cells.Bone marrow cell mobilizer-recombinant human granulocyte colony
 stimulating factor (rhG-CSF) are used to mobilize bone marrow stem cells to peripheral blood,
 and to increase the number of peripheral blood stem cells, especially endothelial progenitor
 cells, so as to treat ischemic diseases using transplanted autologous stem cells, which is
 called autologous stem cell transplantation. Nevertheless, in animal and human experiments,
 the proliferation potential and influencing factors of mobilized bone marrow stem cells and
 peripheral blood endothelial progenitor cells in ischemic tissue require further
 investigations.

 Autologous stem cell transplantation is to transplant stem cells in muscles of ischemic limb,
 so that new capillaries improve and restore lower limb blood flow, aiming to treat lower limb
 ischemia. Endothelial progenitor cells and hematopoietic stem cells are thought to come from
 a common ancestor, and called blood vessel stem cells. They express immature stem cell
 markers CD34 and CD133. Endothelial progenitor cells migrated from bone marrow to peripheral
 blood will gradually lose CD133, and the disappearance of CD34 is relatively slow. A large
 number of basic and clinical studies have confirmed that the transplantation of CD34+ cells
 can promote angiogenesis in ischemic limbs of experimental animals (partial patients).

 Adverse events

 1. To record adverse events, including fever, infection, lower limb pain and ulcers, during
 follow-up at out-patient clinic.

 2. If severe adverse events occur, investigators will report details, including the date of
 occurrence and measures taken to treat the adverse events, to the principle investigator
 and the institutional review board within 24 hours.

 Data collection, management, analysis, open access

 1. Data collection: Case report forms will be collected and processed using Epidata
 software (Epidata Association, Odense, Denmark), collated, and then recorded
 electronically using a double-data entry strategy.

 2. Data management: The locked electronic database will be accessible and locked only by
 the project manager. This arrangement will not be altered. The Qingdao No. 9 People's
 Hospital, China will preserve all of the data regarding this trial.

 3. Data analysis: A professional statistician will statistically analyze the electronic
 database and will create an outcome analysis report that will be submitted to the lead
 researchers. An independent data monitoring committee will supervise and manage the
 trial data, ensuring a scientific and stringent trial that yields accurate and complete
 data.

 4. Data open access: Anonymized trial data will be published at www.figshare.com.

 Statistical analysis

 1. Statistical analysis will be performed using SPSS 19.0 software (IBM, Armonk, NY, USA)
 and will follow the intention-to-treat principle.

 2. Normally distributed measurement data will be expressed as means, standard deviation,
 minimums and maximums. Non-normally distributed measurement data will be expressed as
 the lower quartile (q1), median and upper quartiles (q3). Count data will be expressed
 as a percentage.

 3. Kolmogorov-Smirnov test will be used to determine normality of measurement data. If data
 obey the normal distribution, ABI, number of microvessels in the lower limb muscles and
 number of VEGF-immunoreactive cells will be compared with two-sample t-test between the
 two groups. Intragroup difference of above indicators will be compared using paired
 t-test before and 6 months after transplantation. If data do not obey the normal
 distribution, intergroup and intragroup data will be compared using Mann Whitney U test
 and Wilcoxon signed-rank test. The incidence of adverse reactions will be compared with
 Fisher's exact probability test between the two groups 6 months after transplantation.

 4. The significance level will be α = 0.05.
Inclusion Criteria:

 -  Elderly patients with arteriosclerosis obliterans (Rutherford classification of class
 5: slight tissue defect; class 6: tissue ulceration, gangrene)

 -  Diabetic foot, Wagnar classification of class 315: deep ulcer, often affecting bone
 tissue, with deep abscess or osteomyelitis

 -  Age range: 50-60 years

 -  Irrespective of gender

 -  Unilateral limb lesions

 -  Total occlusion of main blood vessels

 -  No outflow tract in distal arteries

 -  Cannot perform open surgery and endovascular surgery

 -  Signed informed consent

 Exclusion Criteria:

 -  Moderate to severe liver and kidney dysfunction

 -  Malignant tumor

 -  Hematological system diseases

 -  Rheumatic immune system diseases

 -  Poor stimulating effect on colony stimulating factor

 -  Thromboangiitis obliterans

 -  Severe hyperthyroidism

 -  Endocrine and metabolic diseases such as severe hypothyroidism