Differential vascular cell adhesion molecule-1 expression and superoxide production in simulated microgravity rat vasculature.

Exposure to microgravity leads to orthostatic intolerance in astronauts and differential vascular structural and functional adaptations have been implicated in its occurrence. The present study tended to clarify the characteristics of vascular inflammation and oxidative stress in hindlimb unweighting (HU) rat vasculature. Male Sprague-Dawley rats were randomly divided into control (CON) and hindlimb unweighting (HU) groups. Three weeks later, immunohistochemistry was used to localize the expression of vascular cell adhesion molecule-1 (VCAM-1) and laser scanning confocal microscope were used to detect superoxide production. Immunohistochemical results revealed positive staining of VCAM-1 on endothelial cells in HU rat basilar and carotid arteries compared with CON, but not in abdominal aorta and femoral arteries. Meanwhile, HU increased O2·- levels in all the layers of basilar and carotid arteries from HU rat but not in abdominal aorta and femoral arteries from HU rat. These data suggested that differential expression of VCAM-1 and O2·- production were concomitant with the vascular adaptations to simulated microgravity and whether they participate in vascular structure and function remodeling merits further investigation.

Introduction

When humans are exposed to microgravity during spaceflight, fluids shift toward the head and normal pressure gradient is eliminated. These marked hemodynamic changes occur rapidly on exposure to microgravity and could induce subsequent cardiovascular adaptations, which will lead to orthostatic intolerance in astronauts when they come back to the earth with 1 G gravity. Hindlimb unweighting (HU) rat is a widely used and reliable model to simulate the physiological effects of microgravity on humans. As reviewed by Zhang (2001[40]), simulated microgravity induces upward and downward regulations in the structure and function of the cerebral and hindquarter arteries in HU rat. The cerebral arteries showed vascular hypertrophy, enhanced vasoconstriction (Zhang et al., 2000[41]; Zhang, 2001[40]) and impaired endothelium-dependent relaxation (Prisby et al., 2006[24]); however, the abdominal aorta and femoral arteries showed vascular hypotrophy, attenuated vasoconstriction and endothelium-dependent relaxation (Ma et al., 2000[18]; Zhang, 2001[40]). The endothelium-dependent nitric oxide (NO) (Siamwala et al., 2010[28]; White et al., 2010[33]) and ion channel mechanisms (Xie et al., 2005[38], 2010[37]) have been indicated in vascular adaptation to simulated microgravity. Although much progress has been made in the past decade, the underlying mechanism of vascular adaptations to microgravity remains to be established.

Vascular wall inflammation has been regarded as an initial event of cardiovascular disorders. Vascular cell adhesion molecule-1 (VCAM-1), a cell surface protein expressed by endothelial cells (ECs), mediates lymphocyte recruitment and adherence via interaction with very late antigen-4 (Abdala-Valencia and Cook-Mills, 2006[1]; Cook-Mills, 2002[8]). The lymphocytes release cytokines such as IL-1β, TNF-α, IL-6 which are believed to result in vascular structural remodeling (Mahmud and Feely, 2005[19]), higher vascular reactivity (Jimenez-Altayo et al., 2006[13]). Then whether vascular inflammation contributes to differential vascular adaptations to microgravity? Therefore, the first purpose of the present study was to explore the characteristics of VCAM-1 expression in HU rat vasculature.

The term “oxidative stress” describes the oxidative injury caused by reactive oxygen species (ROS), including superoxide anions (O2·-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), NO and peroxynitrite (ONOO−). ROS can oxidize lipoproteins, limit the vascular availability of NO, and promote vascular expression of cytokines and adhesion molecules (Koh et al., 2009[15]). These radicals play the pathophysiological roles both in the maintenance of steady vessel wall conditions and in the vascular reactivity to altered blood flow or pressure settings (Lehoux, 2006[16]). O2·- has been indicated in regulating vascular inflammation, reactivity and nitric oxide synthase (NOS) expression in hypertensive rats (Vaziri et al., 2000[31]). An increasing body of evidence suggests that oxidative stress is involved in the pathogenesis of many cardiovascular diseases, including hypercholesterolemia, atherosclerosis, hypertension, heart failure and diabetes. Then whether vascular oxidative stress contributes to differential vascular adaptations to microgravity? Therefore, the second purpose of the present study was to explore the characteristics of O2·- production in HU rat vasculature.

Materials and Methods

Animal treatment

The protocols and procedures used in this study were in accordance with Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences. 13-week-old male Sprague-Dawley rats were randomly assigned to 3-week hindlimb unweighting rat (HU) and control (CON). The head-down hindlimb unweighting model described in detail previously (Ma et al., 2003[17]) was used to simulate microgravity in rats. The rats in HU group were maintained in about 30o head-down tilt position and housed individually under a 12:12 h light-dark circle with food and water available ad libitum at 24 ± 1 °C.

Tissue preparation

After 3 weeks of treatment, rats were anesthetized with pentobarbital sodium (40 mg/kg, ip) and dissected. Basilar arteries, common carotid arteries, abdominal aorta and femoral arteries were rapidly removed and placed in Krebs solution consisting of (in mmol/L): NaCl, 118.3; KCl, 14.7; KH2PO4, 1.2; MgSO4·7H2O, 1.2; CaCl2·2H2O, 2.5; NaHCO3, 25; dextrose, 11.1; and EDTA, 0.026; pH 7.40. Parallel weight of soleus muscle-to-body mass ratio was used to test the efficacy of simulated microgravity. The arteries were divided into two parts. The artery segments for immunohistochemistry were fixed in 4 % paraform, and in addition, the artery slice from another part were prepared for detecting vascular O2·- levels.

Immunohistochemical localization

Arterial segments from HU and CON rats to be examined for VCAM-1 expression were immediately frozen in Tissue-Tek O.C.T. embedding medium. 8 μm thick tissue sections were mounted on polylysine-coated slides. After preincubated with 20 % normal bovine serum for 30 min at room temperature to block the nonspecific antigens, the sections were incubated with rabbit polyclonal antibody against rat VCAM-1 (1:50, Santa Cruz Biotechnology) at 4 °C overnight in a moist chamber. The sections were incubated with a biotinylated secondary antibody (1:700, Santa Cruz Biotechnology) in PBS for 2 h at 37 °C after washing with PBS. After another three washes in PBS, the horseradish peroxidase was developed with 3,3-diaminobenzadine (DAB) (Roche Diagnostics, Mannheim, Germany) as the chromogen substrate. The sections were rinsed, dehydrated in ethanol, cleared in xylene, and mounted.

Detection of vascular O2-levels

Dihydroethidium (DHE) is the most popular probe used to detect O2·- levels in vascular tissues and has been described in detail before (Münzel et al., 2002[22]; Zanetti et al., 2005[39]). Arteries for investigation were harvested carefully from the rats and immediately frozen in Tissue-Tek O.C.T. embedding medium. 30 μm thick frozen sections were prepared, and then were stained with 10 μmol/L DHE (Molecular Probes). Laser scanning confocal microscopic images were obtained after incubation in a light-protected humidified chamber at 37 °C for 30 minutes.

Drugs and reagents

All drugs and chemicals were obtained from Sigma Chemical (St. Louis, MO). Rabbit polyclonal anti-rat VCAM-1 was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was the product of Invitrogen and dihydroethidium was from Molecular Probes.

Results

General data

The body weight of the rats for investigation increased steadily during the three weeks. The significant lower soleus muscle mass (HU 82.63 ± 4.51 mg; CON 165.01 ± 6.58 mg, P < 0.001) and soleus muscle-to-body mass ratio (CON 0.40 ± 0.01 mg/g; HU 0.21 ± 0.01 mg/g, P < 0.001) confirmed the efficacy of simulated microgravity and the reliability of animal model used in this study.

VCAM-1 expression in rat arteries

The representative expression of VCAM-1 in arteries is shown in Figure 1(Fig. 1). VCAM-1 protein was undetectable in artery segments of basilar artery, common carotid artery, abdominal aorta and femoral artery from CON group (Figure 1A, C, E, G(Fig. 1)). However, strong staining localized to ECs surface in basilar artery (Figure 1B(Fig. 1)) and common carotid artery (Figure 1 D(Fig. 1)) revealed a significant increase expression of VCAM-1; but it was not the case in abdominal aorta and femoral arteries from HU rats (Figure 1 F, H(Fig. 1)).

Figure 1

Representative photomicrographs of immunohistochemical examination for VCAM-1 expression in basilar artery (A, B), common carotid (C, D), abdominal aorta (E, F) and femoral arteries (G, H) from CON (A, C, E, G) and HU (B, D, F, H) rat (×400).

No staining is detectable in rat basilar artery (A), common carotid (C), abdominal aorta (E) and femoral artery (G) from CON and abdominal aorta (F) and femoral artery (H) from HU. VCAM-1 protein expression in rat basilar and common carotid artery from HU was localized predominantly to the endothelial cells.

Superoxide production in rat arteries

Superoxide levels were shown in Figure 2(Fig. 2), weak O2·- signals were detected by laser scanning confocal microscope in basilar (Figure 2A(Fig. 2)), carotid arteries (Figure 2C(Fig. 2)), abdominal aorta (Figure 2E(Fig. 2)) and femoral arteries (Figure 2G(Fig. 2)) from CON rats. Compared with CON, intense O2·- levels were found in all layers of the basilar (Figure 2B(Fig. 2)) and carotid arteries (Figure 2D(Fig. 2)) from HU rats. However, less generation of O2·- was found in the abdominal aorta (Figure 2F(Fig. 2)) and femoral arteries (Figure 2H(Fig. 2)) from HU rats compared with CON.

Figure 2

Representative fluorescence photographs of rat basilar artery (A, B), common carotid artery (C, D), abdominal aorta (E, F) and femoral arteries (G, H). A, C, E, G are from CON rat and B, D, F, H are from HU rat. Arteries were labeled with the oxidative dye DHE, which would react with O2·- to form ethidium and produced a red fluorescence. Original magnification: ×400.

Discussion

The major findings of the present study were that 3-week simulated microgravity induced by hindlimb unweighting resulted in increased VCAM-1 expression and superoxide production in cerebral and carotid arteries but not in abdominal aorta and femoral arteries.

Chronic inflammation in the vasculature is a pathogenic feature of atherosclerosis and cardiovascular disease. Although laboratory evidence found the increased expression of E-selectin or intercellular cell adhesion molecule-1 (ICAM-1) but not VCAM-1 in human umbilical vein ECs when exposed to simulated microgravity (Romanov et al., 2001[26]), evidence from long-term gravity vector demonstrated the expression of ICAM-1, E-selectin and VCAM-1 on cultured ECs (Buravkova et al., 2005[4]). VCAM-1 is also referred to inducible cell adhesion molecule CD106 with a molecule weight of 100-110 kDa. Hemodynamic stress (Chappell et al., 1998[5]; Sung et al., 2007[29]) and angiotensin II (Savoia and Schiffrin, 2006[27]) modulates the expression of VCAM-1. As indicated by Wilkerson and associates (Wilkerson et al., 1999[35]) the mean arterial pressure of the aorta increased nearly 21 mm Hg after 2-week simulated microgravity. Meanwhile, the content of angiotensin II (Ang II) and its receptors, the key components of rennin-angiotensin system (RAS), increased in 4-week HU rat cerebral and carotid arteries and decreased in femoral arteries (Bao et al., 2007[2]; Meng et al., 2002[21]). Based on the above mentioned, we hypothesized that the expression of VCAM-1 might increase in HU rat cerebral and carotid arteries. As expected, 3-week HU upregulated the expression of VCAM-1 in rat basilar and common carotid artery ECs, but not in abdominal aorta and femoral artery ECs.

Ang II, the main effector of the RAS, is one of the major mediators of vascular remodeling in cardiovascular diseases. Ang II mediates many of its physiological and pathophysiological actions by stimulating formation of intracellular ROS, which plays important roles in modulating inflammatory reactions (Cheng et al., 2005[6]; Benigni et al., 2010[3]; Kaneto et al., 2010[14]). ROS are implicated at virtually every stage in the inflammatory response, including vascular permeability, leucocyte adhesion and transmigration, chemotaxis, cell growth and fibrosis (Harrison et al., 2003[12]). NAD(P)H oxidase, mitochondrial electron transport chain, uncoupled NOS, cytochrome P450 monooxygenase and xanthine oxidase have been demonstrated the important sources of ROS in the vasculature and NAD(P)H oxidase has been indicated a major one of the wide sources (Clempus and Griendling, 2006[7]). Ang II induces activation of vascular NAD(P)H oxidase and increases expression of NAD(P)H oxidase subunits (Cheng et al., 2005[6]). In the present study, 3-week hindlimb unweighting increased the production of O2• - in rat basilar and carotid arteries of all the layers, which might be associated with the activation of RAS in HU rat vasculature. Intracellular oxidative stress in vascular cells activates NF kappaB to stimulate transcription of proinflammatory genes, which results in synthesis of protein products such as cell adhesion molecules, cytokines, and chemokines (Pueyo et al., 2000[25]). In transgenic rats, increased Ang II type I (AT1) receptor/NADPH oxidase activation/ROS contributes to vascular endothelial dysfunction and inflammation (Wei et al., 2007[32]). Although we cannot conclude the causation between vascular inflammation and oxidative stress in the settings of simulated microgravity till now, the positive VCAM-1 expression and increased O2• - production in 3-week HU rat cerebral and carotid arteries that we found in the present study merits further investigation.

NO modulates vascular tone, inhibits platelet function, prevents adhesion of leukocytes, and reduces proliferation of the intima (Förstermann, 2010[10]). Endothelial dysfunction characterized by an enhanced inactivation and/or reduced synthesis of NO occurs throughout the cardiovascular disease process. This may be partly attributed by decreased bioactive NO secondary to increased O2• - production by inactivating NO to form ONOO-, which in turn, uncouple endothelial NOS to become a dysfunctional O2• - generating enzyme that contributes to vascular oxidative stress (Marchesi et al., 2009[20]). Inflammation and oxidative stress contribute to endothelial dysfunction while endothelial dysfunction promotes oxidative stress and inflammation (Koh et al., 2009[15]). Localized inflammatory reaction, as well as enhanced local oxidative stress, contributes to abnormal endothelium-dependent relaxation at both coronary conduit and resistance arteries (Pendyala et al., 2009[23]). The activation of the NF kappaB inflammatory pathway by angiotensin II-induced ROS generation contributed to systemic inflammation and endothelium dysfunction in salt-sensitive hypertension (Zhou et al., 2010[42]). TNF-α and IL-1β inhibits the NO-dependent component of endothelium-dependent relaxation by increasing levels of O2·- and the ability of O2·- to inactivate NO (Wimalasundera et al., 2003[36]). However, IL-10 limits Ang II-mediated oxidative stress and vascular dysfunction both in vitro and in vivo (Didion et al., 2009[9]) and normalizes blood pressure and endothelial function in pregnancy-induced hypertensive rats (Tinsley et al., 2010[30]). The differential adaptations to simulated microgravity in rat vasculature has been reviewed by Zhang (2001[40]), and enhanced vasoconstriction and impaired endothelium-dependent relaxation in HU rat cerebral and carotid arteries have been demonstrated in previous studies (Geary et al., 1998[11]; Wilkerson et al., 2005[34]). Although NO-dependent mechanism (Siamwala et al., 2010[28]; White et al., 2010[33]) has been indicated, the underlying mechanism of endothelium-dependent relaxation in the settings of simulated microgravity remains unclear. Since NO plays a pivotal role in angiogenesis (Siamwala et al., 2010[28]) and early changes (3-day HU) in vasoreactivity (White et al., 2010[33]), and differential VCAM-1 expression and O2·- production existed in HU rat arteries, whether they participate in vascular adaptations to simulated microgravity by influencing NO merits further investigation. In addition, whether vascular inflammation and oxidative stress participate in ion channel remodeling should also be clarified.

In conclusion, we observed the differential VCAM-1 expression and O2·- production in HU rat vasculature and further evidence should be provided to clarify whether they participate in vascular remodeling. In addition, whether anti-inflammation and antioxidation treatment could reverse abnormal vascular response to agonists should be clarified.