Alzheimer′s disease (AD) is an irretrievable neurodegenerative disease that causes dementia in the elderly. ¹ The etiopathogenesis of AD is still unclear. Recently, new concepts have emerged regarding the role of endothelial cells (ECs), vascular disease, and oxidative stress in the pathogenesis of AD and mechanisms that contribute to these events. ²,³ Overproduction of reactive oxygen and NO were observed in many neurodegenerative disorders. ⁴,⁵

Endothelial dysfunction has been linked with many acute and chronic neuroinflammatory diseases. ⁶ Many stimuli can induce apoptosis in ECs in vitro, proposing that endothelial apoptosis is the main mechanism in CNS vascular injury, leading to diminished barrier, immune cell penetration of the CNS, and proceeding inflammation. ⁷

In circulation, ECs affect blood vessels, especially in the brain. It is newly transpired that endothelial dysfunction and vascular disease is related on the role of this cell type. ⁸

In brain, functional changes in ECs contribute to reductions in resting blood flow (hypoperfusion), impairment of vasodilator responses, and subsequent cellular injury. ⁹,¹⁰,¹¹,¹² Identification of endogenous molecules and pathways that protect the vasculature may result in targeted approaches to prevent or slow the progression of vascular disease that causes or contributes to the vascular component of dementia and AD. ¹¹

Activation of the rennin-angiotensin system plays a prominent role in vascular dysfunction. Some clinical studies shows that angiotensin-converting enzyme inhibitors (ACE-I), which has clinically been widely used as an anti-hypertensive agent, reduces the incidence of dementia or slows down the rate of cognitive decline in patients with hypertension. ¹³,¹⁴ Ohrui et al. ¹⁵ showed that active ACE-I, but not non-centrally active ACE-I, could slow down the rate of cognitive decline in mild-to-moderate AD patients. ¹⁶

As regards to beneficial effects of ACE-I on endothelial dysfunction ¹⁶,¹⁷,¹⁸ and endothelial apoptosis, the same can be observed in many inflammatory and vascular injury disorders. This is the first study that presents direct experimental evidence associating endothelial apoptosis as a basis of AD pathogenesis and response to an ACE-I therapy.

The study was conducted with the collaboration of the Departments of Physiology, Applied Physiology Research Center, and Neurology Outpatient Department of Al-Zahra hospital, Isfahan University of Medical Sciences between July 2010 and June 2011. A complete explanation of the study was given to each patient and written informed consent was received from all patients. The study protocol was reviewed and approved by the ethics in Research Committee, Isfahan University of Medical Science.

Patients

In this study, 10 patients with AD and 10 healthy controls (age- and sex-matched healthy subjects) were recruited. Diagnosis of AD was based on the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and AD and Related Disorders Association. ¹⁹ Patients with a history of drug abuse, chronic systemic diseases such as diabetes mellitus, hypertension, coronary heart disease, cigarette smoking, alcohol abuse, or acute illness, severe head injury, or seizure disorders, and who were treated with electroconvulsive therapy, major depression, cerebrovascular disease, intoxication, metabolic abnormalities, and dementia caused by diseases other than AD were not included in the study.

Sample collection and preparation

Peripheral venous blood from ADs was sampled into serum tubes. Then serum was centrifuged within 30 min to reduce platelets and stored at -80°C for further analysis; all measurements were performed at the same time.

Cell culture

Human umbilical vein ECs (HUVECs) (National Cell Bank of Iran affiliated with the Pasteur Institute, Tehran, Iran) were cultured in endothelial basal medium (EBM) supplemented with gentamicin, amphotericin B, and 10% fetal calf serum (FCS) until the 3 ^rd passage before the experiments was performed.

For evaluation effects of enalapril on HUVECs treated with sera of AD, we arranged different groups; in the first group, HUVECs were only treated by sera from AD for 24 hours, in the second group, HUVECs were treated by 50 μM enalapril (dissolved in 0.9% NaCl ²⁰,²¹ ) for 24 h, and then sera from AD was added to these cells for another 24 h. In the third group, HUVECs were exposed in the sera of AD for 24 h and then 50 μM enalapril was added to these cells for another 24 h. In the fourth group, HUVECs were treated by sera from healthy individuals for 24 h.

Apoptosis analysis

The rate of apoptosis in HUVECs was evaluated by flowcytometry and Cell-Death Detection kit. For each treatment, a total number of 10 ⁵ cells were washed with ice-cold PBS once and were stained with annexin-propidium iodide (PI) as follows: Cells (10 ⁵ /ml) were incubated with 1 μl annexin V-fluorescein isothiocyanate and 0.5 μl PI (10 mg/ml) in binding buffer (10 mM HEPES, pH 7.4; 150 mM NaCl; 5 mM KCl; 1 mM MgCl ₂ ; 1.8 mM CaCl ₂ ). Subsequently, the cells were analyzed by fluorescence-activated cell sorting (FACScan, Becton-Dickinson). Apoptotic cells were designated as annexin-V ⁺ /PI⁻ cells. Data were analyzed by Cell Quest software. As an additional measure of apoptotic cell death, we assessed the formation of histone-associated DNA fragments by the Cell-Death Detection ELISA kit from Roche (Basel, Switzerland) ²¹ as previously mentioned or according to the manufacturer instruction. ²²

NO metabolite (NO ₂ ) measurement

NO ₂ , an important NO metabolite in culture supernatants, was determined using the Griess reaction (Parameter TM, total NO Assay kit, R and D Systems, USA) according to the manufacturer′s instructions. Briefly, in this assay, NO ₂ is detected colorimetrically as an Azo dye product of the Griess Reaction. The Griess Reaction is based on the two-step diazotization reaction in which acidified NO ₂ produces a nitrosating agent, which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophore azoderivative that absorbs light at 560 nm wavelength. Values were calculated using a standard curve using a standard curve produced with sodium NO ₂ . ²²

Statistical analysis

The data were reported as mean ± SE (standard error of mean). One way analysis of variance (ANOVA), followed by the Tukey′s post hoc test was used for data analysis. All experiments were repeated in three independent replicates. P value of less than 0.05 was considered significant. Statistical analyses were performed using SPSS version 16.

A 24-h treatment of HUVECs with the sera of untreated AD resulted in significantly greater apoptosis than in healthy controls as measured by flow cytometry and Cell-Death Detection Kit (P < 0.05) Figure 1. There were no significant differences in the apoptosis rates of HUVECs between patients with AD.Figure 1

Determination of apoptosis induction in human umbilical vein endothelial cells after exposure with sera from Alzheimer′s disease patients vs. the control group. Apoptosis rates of HUVECs were measured after exposure to media containing 10% serum of patients with AD or the control group for 24 h. Assessment of suppression of AD serum-induced apoptosis by pre and post-treatment of enalapril (50 μM) for 24 h with flow cytometry (the number of apoptotic cells were designated as annexin-V+/PI−/105 cells) (a) a; P < 0.001 and Cell-Death Detection Kit (b), a; P < 0.05 vs. healthy, b, ab; P < 0.05 vs. patients. Enalapril (50 μM) was added to HUVECs 24 h before and after treating with sera from AD patients, the concentration (μm/L) of nitrite in the culture medium was determined. a; P < 0.05 vs. healthy individuals and b; P < 0.05 vs. patients (c)

In this study, we discovered that NO ₂ concentration and apoptotic measurements were significantly higher in the HUVEC media treated by AD serum as compared with the control, and elevation levels of dissolved NO ₂ /NO ₃ metabolite was significantly reduced by co-incubation of an ACE-I both in the pretreatment and post-treatment groups of serum. However, for apoptotic markers, this reduction occurred only when we added ACE-I before treatment of HUVECs with AD′s serum.

Etiopathogenesis of AD, which leads to demientia, is still unclear. Several studies suggested a possible role of oxidative stress in the pathogenesis of AD. ²³,²⁴ In our experimental model, apoptotic measurements in AD were significantly high in comparison with several studies that indicated that apoptosis might contribute to onset and progression of AD. P53 protein plays a part in neuronal apoptosis in the brain of these patients. ²⁵

Furthermore, in cultures of neurons and astrocytes of human and rat as well as in peripheral blood lymphocytes and brain of patients with AD, increasing in the level of p53 protein has been observed. ²⁶,²⁷

It is also believed that inflammatory cytokines in AD, such as TNF-α, are stimulated by activated microglia, which cause increase of oxidative stress and markers of inflammation in AD patients as well as nitrate generation. ²⁷ Elevated NO ₂ concentration in our study could explain that overproduction of reactive oxygen and nitrogen species occurs in neurodegenerative disorders including AD. In the nervous system, NO seems to have both neurotoxic and neuroprotective properties. ²⁷

This NO can derive from overactivation of constitutive isoform of the enzyme NOS (neuronal NOS) or from the expression of inducible isoform of NOS. ²⁷ We believe that evidence of this process could be detectable by using plasma measures and increased level of NO ₂ concentration in treated HUVECs by AD′s serum. In a previously reported study, this hypothesis has been confirmed that there is an increase in serum NO levels in AD patients as compared to that in controls. ²⁸

We used cultured ECs from HUVEC. In these cells, expression of ACE has been shown. ²⁹ The activation of renin-angiotensin system in the brain of patients with AD has been shown in previous reports. ³⁰,³¹,³² Certain drugs of ACE-I may decline the rate of cognitive deterioration in patients with AD. ³¹ Binding of captopril, a kind of ACE-I to endothelial ACE may results in a "site-specific" potentiate in antioxidant defenses. This property of ACE-I has been demonstrated in vitro33 and in vivo. ³⁴,³⁵ ACE-I attenuates oxidative stress-induced EC apoptosis via p38 MAP kinase inhibition. ³⁵

In our study, pretreatment with enalapril could decline both apoptotic measurement and NO ₂ concentration. It seems that enalapril scavenged free radicals and peroxynitrate that are induced in AD serum. The prophylactic treatment of ACE-I in some disease, like in migraine, has been proven. ²²

Our study revealed that enalapril can possibly decline elevated levels of NO in serum and apoptotic measurement that are presented in probable AD patients. There are evidences confirming beneficial effects of ACE-I for AD prophylaxis. ³⁶ However, the role of the elevation of NO level of serum on AD pathogenesis is unclear, and it requires further investigation. Also, in this study, we demonstrated increased apoptosis of HUVECs following incubation of these cells with sera from AD patients and provided evidence supporting a direct stabilizing effects of enalapril on the endothelium.

This work was founded by Grant No. 289279 from the deputy for neurosciences Research, University of Medical Sciences and Isfahan, Iran.

We are grateful to all of the patients who helped in the progression of our project.