# Effect of Lower Extremity Bypass Surgery on Inflammatory Reaction and Endothelial Dysfunction in Type 2 Diabetic Patients

Authors: Pei-Hsuan Tsai; Jun-Jen Liu; Szu-Yuan Chou; Yao-Chung Chang; Sung-Ling Yeh
Journal: Mediators of Inflammation
 (2009)

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## Abstract

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia and dyslipidemia. The abnormalities in nutrient metabolism and elevated inflammatory mediators resulting from DM lead to impairment of wound healing and vulnerability to infection and foot ulcers. Diabetic lower limb ischemia often leads to limb necrosis. Lower extremity bypass surgery (LEBS) is indicated to prevent limb loss in patients with critical leg ischemia. This study investigated the alteration of inflammatory and endothelium dysfunction markers before and after LEBS in DM patients. Twenty one type 2 DM patients with LEBS were included. Blood was drawn before and at 1 day and 7 days after surgery in the patients. Plasma soluble cellular adhesion molecule levels and blood leukocyte integrin expressions were measured. Also, plasma concentrations of endothelin-1 and nitric oxide were analyzed to evaluate the vascular endothelial function. The results showed that there were no significant differences in plasma cellular adhesion molecules, endothelin-1 and nitric oxide levels, nor did any differences in leukocyte integrin expressions before and after the operation. These results suggest that the efficacy of LEBS on alleviating inflammatory reaction and improving endothelial function in DM patients was not obvious.


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## Body

## 1. Introduction

Diabetes
mellitus (DM) was  the 4th leading cause of death in Taiwan in 2007. Many diabetic patients have an increased risk of atherosclerosis,
cerebrovascular disease, and peripheral vascular diseases [1]. The abnormalities in nutrient metabolism and elevated inflammatory
mediators resulting from DM lead to impairment of wound healing and vulnerability
to infection and foot ulcers. Diabetic
lower limb ischemia caused by arterial occlusion is the most common foot injury
leads to lower extremity amputation in DM patients [2]. Lower extremity bypass surgery (LEBS) is indicated to prevent limb loss in patients
with critical leg ischemia. Previous studies
revealed that a large reduction in major amputation rates is associated with
the increase of LEBS [3, 4]. However, the efficacy of LEBS on inflammatory
reaction in DM patients has not been evaluated and
the changes in inflammatory mediators before and after the LEBS remain unknown.Endothelial
dysfunction accompanied by upregulated inflammatory mediators is a major
contributing factor to the pathogenesis of diabetic vascular complications [5].
The injured vasculature endothelium
promotes the expressions of cellular adhesion molecules. Overexpressions of cellular adhesion molecule
facilitate leukocyte-endothelial interactions which may aggravate inflammatory
reaction and tissue damage [6]. Previous
study demonstrated that increased levels of plasma soluble adhesion molecule
occur in type 2 patients [7]. Endothelin-1
(ET-1) is a potent vasoconstrictor with
mitogenic property. ET-1 stimulates
vascular smooth muscle cells proliferation, a major step in the development of
atherosclerosis [8]. Nitric oxide (NO) is a
vasodilator produced by endothelial cells. Other key roles of NO include inhibiting platelet aggregation, smoothing
muscle cells proliferation, reducing monocyte adherence, and so forth [9]. Previous study showed that at the onset of
diabetes, the release or response to NO is reduced [10]. Both ET-1 and NO are important mediators in
maintaining vascular functions. Since LEBS
increases blood flow to tissues that may carry
oxygen and nutrients
to the lower extremities and improve
the healing of the tissues [11], we hypothesized that the inflammatory process and vascular dysfunction
are attenuated in DM patients undergoing LEBS. Therefore, the aim of this study was to investigate whether reperfusion
of the lower extremities may improve the inflammation and endothelial
dysfunction in DM patients.


## 2. Subjects and Methods

### 2.1. Subjects

This study was conducted from April
to December 2006 at Taipei Medical University-affiliated Wan Fang
Hospital. Twenty one type 2 DM patients with
severe diabetic lower limb ischemia and underwent LEBS were enrolled in the
experimental group. The diabetes
duration was 6–30 years, with a mean of 18.2 years. Insulin and combined therapy if necessary were used to control blood
glucose to within a range of 106–259 mg/dL. No leg infection was observed in LEBS patients, possibly because the
operation was successful and the antibiotics used after the surgery. The protocol was approved by the hospital ethics
committee, and all the patients gave their informed written consent prior to
their participation in this study.


### 2.2. Blood Sampling

Blood
samples were taken from each patient before and at 1 day and 7 days after the LEBS in
DM patients. Ten milliliters of blood
were drawn after 12 hours of fasting, placed in tubes containing ethylene
diaminetetraacetic acid. Fresh blood 	 samples were collected for the analysis of leukocyte CD11a/CD18 and CD11b/CD18 expressions. Plasma samples obtained from whole blood
centrifugation at 3000 rpm for 10 minutes were stored at−80°C until further analysis.


### 2.3. Measurements and Analytical Procedures

Total
cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein
cholesterol, triglyceride, and creatinine were analyzed by an autoanalyser (Hitachi 7170, Tokyo, Japan). Blood HbA1C was measured using a commercial
kit (Helena BioSciences, sunderland, UK). Procedures followed the manufacturer
instructions.


### 2.4. Measurements of Plasma Et-1, No, Sicam-1, Svcam-1, and C-Reactive Protein Concentrations

Concentrations of plasma ET-1, soluble
intracellular adhesion molecule (sICAM)-1,
soluble vascular cell adhesion molecule (sVCAM)-1,
and C-reactive protein (CRP) were
measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D systems,
Minneapolis, MN, USA). Concentrations of NO2−/NO3− were determined with a commercial kit (R&D systems). Procedures followed the manufacturer
instructions. The minimum detectable
dose of ET-1, sICAM-1, sVCAM-1, and CRP were 0.064 pg/mL, 0.35 ng/mL, 0.6 ng/mL,
1 ug/ml, respectively, for NO2−/NO3− was
0.25 uM.


### 2.5. Analysis of Lymphocyte Cd11a/cd18 and Polymorphoneuclear Neutrophil Cd11b/cd18 Expressions

One hundred microliters of fresh blood was incubated with
10 uL fluorescent isothiocynate (FITC)-conjugated
mouse monoclonal antihuman CD11a and phycoerthrin (PE)-conjugated mouse
monoclonal antihuman CD18 (Serotec, Oxford, UK) for 15 minutes at 4°C. The proportions of CD11a/CD18 expressed on lymphocytes were analyzed by
flow cytometry (Coulter, Miami, FL). The results are
presented as a percentage of CD11a-presenting
cells in 1 × 105 lymphocytes. To
measure CD11b/CD18 expressions on polymorphoneuclear neutrophils (PMNs), FITC-conjugated
mouse monoclonal antihuman CD11b and PE-conjugated mouse monoclonal antihuman
CD18 (Serotec) were added into 100 μL of fresh
blood. The results are
presented as a percentage of CD11b/CD18 expression in 1 × 105 PMNs. Lymphocytes and PMNs were
gated on the basis of the forward scatter and side scatter profiles and were
analyzed for the expressions of CD11a/CD18
and CD11b/CD18, respectively.


### 2.6. Statistical Analysis

Data are
presented as mean ± SD. All statistical analyses were performed using
SAS software package. The differences among
different time points were determined by one-way analysis of variance.P < .05 was considered statistically significant.


## 2.1. Subjects

This study was conducted from April
to December 2006 at Taipei Medical University-affiliated Wan Fang
Hospital. Twenty one type 2 DM patients with
severe diabetic lower limb ischemia and underwent LEBS were enrolled in the
experimental group. The diabetes
duration was 6–30 years, with a mean of 18.2 years. Insulin and combined therapy if necessary were used to control blood
glucose to within a range of 106–259 mg/dL. No leg infection was observed in LEBS patients, possibly because the
operation was successful and the antibiotics used after the surgery. The protocol was approved by the hospital ethics
committee, and all the patients gave their informed written consent prior to
their participation in this study.


## 2.2. Blood Sampling

Blood
samples were taken from each patient before and at 1 day and 7 days after the LEBS in
DM patients. Ten milliliters of blood
were drawn after 12 hours of fasting, placed in tubes containing ethylene
diaminetetraacetic acid. Fresh blood 	 samples were collected for the analysis of leukocyte CD11a/CD18 and CD11b/CD18 expressions. Plasma samples obtained from whole blood
centrifugation at 3000 rpm for 10 minutes were stored at−80°C until further analysis.


## 2.3. Measurements and Analytical Procedures

Total
cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein
cholesterol, triglyceride, and creatinine were analyzed by an autoanalyser (Hitachi 7170, Tokyo, Japan). Blood HbA1C was measured using a commercial
kit (Helena BioSciences, sunderland, UK). Procedures followed the manufacturer
instructions.


## 2.4. Measurements of Plasma Et-1, No, Sicam-1, Svcam-1, and C-Reactive Protein Concentrations

Concentrations of plasma ET-1, soluble
intracellular adhesion molecule (sICAM)-1,
soluble vascular cell adhesion molecule (sVCAM)-1,
and C-reactive protein (CRP) were
measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D systems,
Minneapolis, MN, USA). Concentrations of NO2−/NO3− were determined with a commercial kit (R&D systems). Procedures followed the manufacturer
instructions. The minimum detectable
dose of ET-1, sICAM-1, sVCAM-1, and CRP were 0.064 pg/mL, 0.35 ng/mL, 0.6 ng/mL,
1 ug/ml, respectively, for NO2−/NO3− was
0.25 uM.


## 2.5. Analysis of Lymphocyte Cd11a/cd18 and Polymorphoneuclear Neutrophil Cd11b/cd18 Expressions

One hundred microliters of fresh blood was incubated with
10 uL fluorescent isothiocynate (FITC)-conjugated
mouse monoclonal antihuman CD11a and phycoerthrin (PE)-conjugated mouse
monoclonal antihuman CD18 (Serotec, Oxford, UK) for 15 minutes at 4°C. The proportions of CD11a/CD18 expressed on lymphocytes were analyzed by
flow cytometry (Coulter, Miami, FL). The results are
presented as a percentage of CD11a-presenting
cells in 1 × 105 lymphocytes. To
measure CD11b/CD18 expressions on polymorphoneuclear neutrophils (PMNs), FITC-conjugated
mouse monoclonal antihuman CD11b and PE-conjugated mouse monoclonal antihuman
CD18 (Serotec) were added into 100 μL of fresh
blood. The results are
presented as a percentage of CD11b/CD18 expression in 1 × 105 PMNs. Lymphocytes and PMNs were
gated on the basis of the forward scatter and side scatter profiles and were
analyzed for the expressions of CD11a/CD18
and CD11b/CD18, respectively.


## 2.6. Statistical Analysis

Data are
presented as mean ± SD. All statistical analyses were performed using
SAS software package. The differences among
different time points were determined by one-way analysis of variance.P < .05 was considered statistically significant.


## 3. Results

The
characteristics of the subjects were presented in Table1. There were no significant differences in
plasma ET-1 and NO levels (Table 2), nor
did any differences in plasma sVCAM, sICAM, and CRP levels before and after the operation
(Table 3). Also, leukocyte CD11a/CD18 and CD11b/CD18 expressions before and after
the operation did not differ in DM patients (Table 4).Table 1
Characteristics of the subjects.
Diabetic patientsAge (yr)70.7±9.0Gender (M/F)10/11Blood glucose (mg/dL)175.9±59.5HbA1C (%)8.7±2.3Total cholesterol (mg/dL)152.9±35.6HDL-C (mg/dL)32.7±13.1LDL-C (mg/dL)86.0±33.2Triglyceride (mg/dL)145.8±81.6Creatinine (mg/dL)3.70±3.44Data are expressed as the mean ± SD. Abbreviations:
HbA1C: Hemoglobin A1C; HDL-C:
High-density lipoprotein-cholesterol; LDL-C: Low-density lipoprotein-cholesterol.Table 2
Plasma endothelin (ET)-1 and nitric oxide (NO) concentrations before and after the surgery.
DaysET-1NO(pg/mL)(umol/L)02.2±0.53.5±1.712.0±0.22.5±0.772.2±0.43.4±1.9Data are
expressed as the mean ± SD.Table 3
Plasma soluble intercellular adhesion
molecule (sICAM), soluble vascular cell
adhesion molecule (sVCAM), and
C-reactive protein (CRP) concentrations
before and after the surgery.
DayssVCAMsICAMCRP(ng/mL)(ug/mL)01406.2±839.7344.3±121.853.4±46.711415.9±722.6332.3±112.164.6±54.971224.9±632.8398.5±126.948.3±45.7Data are expressed as the mean ± SD.Table 4
Expressions of lymphocyte CD11a/CD18 and neutrophil CD11b/CD18
before and after the surgery.
DaysCD11a/CD18CD11b/CD18(%)048.2±18.03.1±2.0142.4±12.02.9±1.3743.5±12.33.5±1.1Data are expressed as the mean ± SD.


## 4. Discussion

This study
evaluated an LEBS-induced change in inflammatory response and endothelial
function. We found that compared with the
preoperative condition, the mediators related to inflammation and vascular
function did not change in type 2 DM patients at early and late stages after
LEBS.Diabetic patients often have endothelial dysfunction and
releasing of endothelins is partly responsible for this. Previous report found that plasma ET-1 levels
are enhanced in patients with poor glycemic control [12] and ET-1 levels were
even higher in DM patients complicated with vascular diseases [13]. A study performed by Schneider
et al. [14] found that diabetic patients taking angiotensin converting enzyme
inhibitors had lower plasma ET-1 levels than patients without, indicating that
medical intervention did improve ET-1 levels. In addition to ET-1, NO is also an important regulatory determinant of
vascular tissue homeostasis. NO plays a
protective role by suppressing abnormal proliferation of vascular smooth muscle
following various vascular interventions such as bypass grafting [15]. In this study, we did not observe differences
in plasma ET-1 and NO levels before and 1 or 7 days after surgery. This result may indicate that LEBS performed
in this study did not, at least in the short run, improve endothelial function
in DM patients.CRP is an inflammatory
marker. CRP levels were correlated with
peripheral artery disease severity in patients undergoing LEBS [16]. ICAM-1 and VCAM-1 are adhesion proteins
synthesized by endothelial cells. Their
expressions greatly increase after stimulation by proinflammatory cytokines [17]. Previous study showed that tissue ICAM-1
levels were positively correlated with blood glucose levels [18]. Adhesive interactions between leukocytes and endothelial
cells are involved in inflammatory or immunologic response mechanisms. Adhesion molecules on endothelial cells are
the ligands of integrins on leukocytes. CD11a
and CD11b/CD18 are members of the leukocyte adhesion molecules β2 integrin. CD11a/CD18 are exclusively
expressed on leukocytes and CD11b/CD18 are abundant in PMNs [19]. In this
study, we analyzed lymphocyte CD11a/CD18 because the function of T-lymphocyte
subsets is important on influencing the type of immunity and the inflammatory
response to infection [20]. CD11b/CD18
expressed by neutrophil is important in mediating neutrophil-endothelial cell
interactions and binding to adhesion molecule on the surface of vascular
endothelial cells [21, 22]. Previous reports revealed that
neutrophil CD11b/CD18 increases in infected patients and is correlated with
microvascular dysfunction [23]. In this study, we did not observe
the difference in leukocyte CD11a/CD18 and CD11b/CD18 expressions before and
after the operation. This result was
consistent with plasma sVCAM-1, sICAM-1, and CRP levels that these inflammatory
proteins did not change after LEBS. These
findings indicate that compared with preoperative state, LEBS did not attenuate
inflammatory reaction in DM patients. Surgery and trauma induce
a generalized state of inflammation. Although LEBS increases blood flow to the
peripheral tissues, surgical injury stimulates the production of endogenous
inflammatory mediators. Besides,
reperfusion of the ischemic tissues may also result in exaggerated inflammatory
response [24, 25], this may make the alteration of inflammatory mediators not so obvious
before and after the surgery.In conclusion, this is a pilot study
to demonstrate that compared with the preoperative condition, no
differences in plasma concentrations of ET-1, NO, and inflammatory mediators were
observed in DM patients after LEBS. This
result suggests that the effect of LEBS on alleviating the inflammatory
reaction and improving endothelial function in DM patients was not obvious.



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*Source: 417301-2009-04-05.xml*