Methods

The inclusion criteria were a previous diagnosis of type 2 diabetes mellitus, documentation of chronic hyperglycemia with hemoglobin A1c of ≥6% at diagnosis, ≥18 years old, either gender, and any race. The patients could be treated with metformin, sulfonylureas, acarbose, and/or supplementary insulin but not with thiazolidinediones or thienopyradines. Exclusion criteria included uncontrolled hypertension (blood pressure >140/90 mm Hg), history of stroke, creatinine >2 mg/dl, and malignancy.

After enrollment, an initial blood specimen was assayed for mature CECs, after which patients began treatment with clopidogrel, 75 mg/day, taken orally for 1 month. The hemoglobin A1c level, body mass index, age, gender, and coincident medication use were also recorded at entry. Clopidogrel was discontinued after 1 month of therapy, at which time a second blood specimen was obtained. No other medication changes were made during the month of treatment. All participants provided written informed consent, and the Committee for Protection of Human Subjects, the institutional review board of New York Medical College, approved the study. All investigations were performed in accordance with the principles of the Declaration of Helsinki, as revised in 2000.

A 15-ml sample of peripheral blood was obtained, and the first 7.5 ml was discarded. Of the remaining sample, 2 ml of blood was diluted with 2 ml of phosphate-buffered saline, 0.1% bovine serum albumin sodium azide, and 20 μl FcR blocking agent (Miltenyi, Gladbach, Germany) and incubated with 100 μl anti-CD146-coated, 45-μm Dynabeads (1.4 × 10 ⁸ coated beads/ml) for 30 minutes at 4°C in a Dynal mixer (Dynal, Lake Success, New York) at 50 rpm. Cells bound to anti-CD146-coupled beads were separated from blood in a Dynal magnet, washed (3 washings using phosphate-buffered saline and 0.1% bovine serum albumin and repetitive mixing for 5 minutes in the Dynal mixer at 4°C), and dissolved in 100 μl buffer. Side-by-side assays were performed with Dynabeads coated with human antibodies against mouse IgG but without anti-endothelial antibody to check for nonspecific binding to the Dynabeads. The cells were mixed with acridine and incubated with 10 μl Ulex (1:1,000) for 2 hours at room temperature, visualized by light and fluorescence microscopy, respectively, and counted in a Nageotte chamber (Brand, Wertheim, Germany). The cells were also stained with anti–von Willebrand factor VIII antibody. CECs were quantitated by counting performed in parallel using both methods ( Figure 1 ).

Morphology of CECs with Dynabeads attached in blood from patients with type 2 diabetes mellitus. Typical CECs captured by Dynabeads coated with anti-CD146 under light (a) or fluorescent microscopy (b) . CECs stained with acridine orange, isolated by Dynabeads, and stained with Ulex europaeus agglutinin 1 lectin.

Nonspecific binding of leukocytes to Dynabeads, presumably by interaction with F _c receptors, has been well documented, and the following steps were taken to avoid it. The cell suspension was flushed vigorously through a 20-μL pipette tip, and citrate was added to the buffer with F _c blocking agent to prevent this problem. Side-by-side assays with M-450 Dynabeads coated with human antibodies against mouse IgG but without anti-endothelial antibody were also performed to check for nonspecific binding to Dynabeads. Isolated cells were stained with antibodies to von Willebrand factor (Boehringer, Mannheim, Germany), CD31 (Binding Site, Heidelberg, Germany), and Ulex europaeus lectin 1 (Linaris, Wertheim, Germany) and with acridine.

Concurrently, EPCs were isolated in a similar fashion using anti-CD34 and, subsequently, anti-CD133. The cells were detached from the beads, suspended in endothelial medium (endothelial growth medium-2), and plated in collagen-coated 6-multiwell plates. The growth medium was changed after 3 days, and the colonies were assessed by light microscopy at 5 days. The lack of hematopoietic contaminants was tested using a fluorescence-activated cell sorter and was negative for CD45.

EPCs were suspended in buffer and centrifuged at 27,000 g for 10 minutes at 4°C. The supernatant was isolated, and the protein levels were determined by immunoblotting with antibodies against phosphorylated Akt and phosphorylated adenosine monophosphate kinase (Cell Signaling Technology, Beverly, Massachusetts) prepared in a 1:5,000 dilution. In brief, 20 μg of supernatant was separated by 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Immunoblotting was performed as previously described. Chemiluminescence detection was performed with the Amersham ECL detection kit (Amersham, Piscataway, New Jersey), according to the manufacturer’s instructions.

Patients functioned as their own controls, with the first specimen obtained at entry compared to the second specimen obtained after 1 month of treatment. We have previously documented a mean of 70 cells/ml of whole blood in patients with type 2 diabetes mellitus compared to 10 cells/ml in normal controls. Assuming a minimum of a 60% decrease in CECs/ml in the experimental group, it was determined that a minimum of 8 subjects would need to be studied to reject the null hypothesis with a power of 0.8. Unpaired Mann-Whitney U testing (2-sided) was used for comparison of cell numbers between blood specimens after the Kruskal-Wallis test had been applied to show significant differences between specimens. Statistical significance between the groups was determined by analysis of variance analysis. p Values ≤0.05 were considered statistically significant. The data are presented as the mean ± SE.

Results

A total of 9 subjects (5 men and 4 women, mean age 55.2 years) were enrolled in the present study. The subjects were recruited from the general medical and endocrine clinics of the Westchester Medical Center (Valhalla, New York), and each had been diagnosed with type 2 diabetes mellitus >2 years previously. The subject characteristics are summarized in Table 1 . Most patients were medicated with metformin, aspirin, and either an angiotensin-converting enzyme inhibitor or an AT ₁ blocker.

Table 1

Patient characteristics

Pt. No.	Age (years)	Gender	Risk		Medications
			D	SH	A	ACEI	AT 1	St	F	E	βB	αB	Su	D4-I	M	I	IM	T	W	T	PPI	S
1	31	Female	0	0	+	+	0	0	0	0	0	0	0	0	0	+	0	0	0	0	0	0
2	42	Female	+	0	+	+	0	+	0	0	0	0	0	0	+	0	+	0	0	0	0	0
3	50	Male	+	+	0	0	0	0	0	0	0	0	0	+	0	0	0	0	0	0	+	+
4	52	Female	+	+	+	0	+	+	0	0	0	0	0	0	+	0	+	0	0	0	0	0
5	59	Male	+	+	+	+	0	+	+	0	+	0	+	0	+	0	0	0	0	0	0	0
6	60	Male	+	+	+	0	0	+	0	0	0	+	+	0	0	0	0	0	0	0	0	0
7	61	Male	+	+	+	+	0	+	0	+	+	0	+	0	0	0	0	0	0	0	0	0
8	65	Male	+	+	+	+	0	+	0	0	+	0	0	0	+	0	0	0	0	0	0	0
9	75	Female	+	+	+	+	0	+	0	0	+	0	0	0	+	+	0	0	+	+	0	0

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