The authors have declared that no competing interests exist.
Conceived and designed the experiments: JJ Y-XQ Z-LJ. Performed the experiments: JJ Y-XQ PZ W-TG Z-QY B-RS Q-PY HK. Analyzed the data: JJ Y-XQ SC Z-LJ. Contributed reagents/materials/analysis tools: JJ Y-XQ Z-LJ. Wrote the paper: JJ Y-XQ SC Z-LJ.
Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.
Vascular endothelial cells (ECs), which form the inner surface of blood vessel wall, serve important homeostatic functions in maintaining the vascular physiological states. EC functional changes, such as abnormal permeability, proliferation, apoptosis, alignment, production of chemotactic molecules, and expression of adhesion molecules, etc., play significant roles in many vascular diseases
In the pathological process of hypertension, cyclic mechanical strain subjected to the arterial wall increases accordingly. Cyclic strain of brachial arteries is about 5% in normal state and can be elevated to 15% in hypertension
To evaluate the mechanism involved in EC functional changes during hypertension, we focus on a novel molecule with potential mechano-sensitivity, Rab28, which was firstly revealed by our previous vascular proteomic study
Rab family is the largest family of small Ras-like GTPase with more than 60 members in human
To evaluate the role of increased Rab28 expression in vessels during hypertension, the cyclic strain loading system was used to mimic the mechanical situation of hypertension in vitro, and to evaluate the role of cyclic strain-modulated Rab28 expression on EC functions. This study provided novel information on the expression, intracellular distribution, and functions of Rab28 in ECs. Understanding of the mechanobiological mechanisms of Rab28 on EC homeostasis will help to define the molecular mechanisms underlying vascular remodeling.
VSMCs and ECs cultured from rat aorta were subjected to normal cyclic strain (physiological, 5% elongation at 1.25 Hz) and high cyclic strain (pathological, 15% elongation at 1.25 Hz) for 24 hours, respectively (
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Rab28 expression of VSMCs was very low in both the static (0% elongation) and the physiological 5% cyclic strain group. While the pathological 15% cyclic strain significantly increased the Rab28 expressions of VSMCs in comparison to the static as well as 5% cyclic strain (
It has been shown that interaction between ECs and VSMCs via paracrine control or direct contact plays a vital role in vascular homeostasis
Angiotensin II (Ang II) is one of the most important vasoactive molecules secreted from vascular tissue in situ and plays key roles in vascular remodeling through paracrine effects during hypertension
Rab28 expression in ECs was also induced by exogenous Ang II stimuli in a concentration-dependent manner (
To evaluate whether the changed expression of Rab28 participated in the modulation of vascular cell functions, the expression of Rab28 was “knocked-down” by target siRNA transfection, and the migration, proliferation and apoptosis of ECs and VSMCs were then analyzed. The results revealed that the suppressed expression of Rab28 in ECs reduced their proliferation, but enhanced apoptosis and migration (
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The induced expression of Rab28 in ECs by the CM from VSMCs subjected to 15% cyclic strain was accompanied by a significant increase of EC proliferation and decrease of apoptosis (
Since all well-studied Rab GTPases are localized in the cytoplasm and related to vesicle traffic
After synchronizing the ECs with serum-free medium for 24 hours, Rab28 mainly existed in the cytoplasm (
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Confocal microscopy revealed the co-existence of Rab28 and NF-κB in the cytoplasm in the synchronized ECs (
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In order to assess the interactions between Rab28 and NF-κB, Rab28 expression was knocked down in ECs. Down-regulation of Rab28 by RNA interference attenuated translocation of NF-κB into the nucleus (
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Long-term hypertension causes vascular remodeling, which is characterized by thickening of vascular media, decreasing of inner-radius-to-thickness ratio, and imbalance of cell proliferation and apoptosis. In the present research, we demonstrated that the pathological cyclic strain up-regulated the expression of Rab28 of ECs via the paracrine effect of VSMCs, which might contribute to the disturbance of EC homeostasis.
Target siRNA transfection significantly decreased Rab28 expression, which down-regulated proliferation and up-regulated apoptosis and migration of ECs, and suppressed migration of VSMCs. The effects of Rab28 on biological behaviors of vascular cells, especially on EC growth and survival, suggest that the modulations of Rab28 expression might participate in vascular remodeling during hypertension.
To prove this hypothesis and evaluate the possible mechanisms by which Rab28 regulates cell functions, the Flexcell cyclic strain loading system was used to simulate the mechanical condition applied to ECs and VSMCs during hypertension
Ang II is one of the most important bioactive molecules secreted from VSMCs in situ. It has been proven that Ang II plays key roles in vascular remodeling during hypertension
Generally, Rab GTPases act as “traffic switches” between organelles, and their cellular distributions are related to their function. Therefore, we investigated the shuttling of Rab28 between cellular compartments in ECs. Cytoplasm-nucleus shuttling, but not cytoplasm-organelles shuttling, of Rab28 is detected in ECs induced by Ang II. It is known that with Ang II stimulation, NF-κB dissociates from the inhibitor of NF-κB (IκB) to become activated
Our results indicate that both Rab28 and NF-κB positively influence EC proliferation, while negatively regulate EC apoptosis and migration. Furthermore, Rab28 might assist the nuclear transport of NF-κB to regulate EC functions, in view of (a) the similar cytoplasm-nucleus translocation of both Rab28 and activated NF-κB, (b) the co-localization of Rab28 and NF-κB in both the cytoplasm and nucleus, (c) co-IP of Rab28 and NF-κB p65, and (d) the roles of the other Rab GTPase family members in intracellular trafficking.
Ang II and NF-κB play important roles in vascular cell proliferation and apoptosis in the presence of mechanical force. The effects of Ang II on cell proliferation are augmented by mechanical stretch in VSMCs of spontaneously hypertensive rat
Pathological cyclic strain loading on VSMC activates the local angiotensin system. VSMC secretes Ang II into the intercellular space (conditioned medium). Ang II induces the expression and translocation of Rab28 in ECs, which aids the NF-κB nuclear transporting and activation. Then the activated NF-kB eventually modulates gene transcription in charge of EC proliferation, apoptosis and migration.
Large molecules (>40 kD) cannot pass through nuclear envelope freely. Their nuclear transport is controlled by the nuclear pore complex (NPC)
Taken together, our results suggest that Rab28 might modulate EC proliferation, apoptosis and migration through its assistance of NF-κB translocation from the cytoplasm into the nucleus. This study has revealed novel information on the expression, intracellular distribution and functions of Rab28. Understanding of the effects of Rab28 on molecular “switches” and biological function will help to define the molecular mechanisms underlying vascular homeostasis and development of vascular pathologies, such as atherosclerosis, thrombosis, hypertension, as well as their clinical complications.
All procedures involving animals conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996), and were approved by the Animal Research Committee of Shanghai Jiao Tong University.
The SD rats were euthanized at the end of the experiments with sodium pentobarbital at 120 mg/kg. After the animal was sacrificed, the thoracic aorta was surgically removed. ECs and VSMCs were isolated from the thoracic aorta of healthy SD rat as previously described
The cells were subjected to cyclic strain provided by a cyclic strain loading system FX-4000 (Flexcell international, Hillsborough, NC, USA), with an elongation magnitude of 5% at a frequency of 1.25 Hz to simulate the physiological vascular environment, and 15% at 1.25 Hz to simulate the hypertensive environment.
ECs in the vessel wall in vivo are adjacent to VSMCs, and there is closely functional interrelation between them. To study the reciprocal paracrine interactions between ECs and VSMCs in the condition of different cyclic strain, the CM of VSMCs subjected to different magnitudes of cyclic strain were transferred to the static ECs, and the CM of ECs subjected to cyclic strains were transferred to the static VSMCs.
Vascular cells were seeded on the 96-well plate after being subjected to cyclic strains and those treated with CM. The cells were incubated with bromodeoxyuridine (BrdU) (Basel, Roche, Switzerland) for 8 hours. The proliferation level of vascular cells was detected with a BrdU antibody (Roche) followed by an enzyme-linked immunosorbent assay (ELISA) protocol. Proliferating cell nuclear antigen (PCNA) detection by Western blot (PCNA antibody. Sigma, St. Louis, MO, USA) was also used to determine cell proliferation.
The cells subjected to cyclic strains and those treated with CM were gently digested from the original plates and re-suspended. The cells were then incubated with Annexin V-FITC (R&D systems, Minneapolis, MN, USA) and propidium iodide (PI). Cell apoptosis level was detected by a flow cytometry (FACS Calibur. Becton Dickinson, Franklin Lakes, NJ, USA). Detection of caspase-3 by Western blot (Caspase-3 antibody. Cell Signaling, Danvers, MA, USA) was also used to assess cell apoptosis.
The CM from the VSMCs subjected to cyclic strains was transferred to 96-well plates. Ang II concentrations in the media were detected with an Ang II EIA kit (Phoenix Pharmaceuticals, Burlingame, CA, USA).
To block the effect of Ang II, ECs were pre-treated with AT1R blockers, Irbesartan and Eprosartan, for 60 min. Then the CM from VSMCs was used to incubate ECs.
To study the effects of Ang II on the Rab28 expression in ECs, exogenous Ang II was added to the static ECs.
To evaluate the role of Rab28 on the functions of vascular cells, the expression of Rab28 was “knocked-down” by target small interfering RNAs (siRNA) transfection. The strands of siRNA for Rab28 were synthesized (GenePharma, Shanghai, China): 5′-GGCAAGAUGUUGGAUAAAUTT-3′, 5′-AUUUAUCCAACAUCUUGCCTT-3′. The siRNA were transfected to ECs with Lipofectamine 2000™ (Invitrogen, Carlsbad, CA, USA).
Rab28 expression was detected by Western blot with anti-Rab28 antibody (Abcam, Cambridge, UK) and the activation level of NF-κB was detected by Western blot with anti-phosphor-NF-κB p65 antibody (Sigma, USA).
Cell migration was detected by using the Transwell system (Costar, USA). Cell proliferation was evaluated by BrdU-ELISA and PCNA expression. Cell apoptosis was evaluated by Annexin V-FITC flow cytometry and caspase-3 expression.
In order to elucidate the mechanism by which Rab28 regulates proliferation, apoptosis and migration of ECs, the intracellular distribution and translocation of Rab28 was determined. ECs were cultured under serum-deprivation, normal growth condition, and Ang II stimulation, respectively. Rab28 protein distributions in ECs under different conditions were studied by immunofluorescence (anti-Rab28 antibodies, Abcam) using a confocal microscope (FV1000. Olympus, Shinjuku, Tokyo, Japan).
Based on the distribution of Rab28 in ECs, Rab28 and NF-κB were double-stained with their respective antibodies. Ang II, 10−6 mol/L, was used to stimulate ECs for 12 hours.
ECs were incubated with 10−6 mol/L Ang II for 1 hour and lysed with cold RIPA lysate on ice. After centrifugation, anti-Rab28 antibody was added to the corresponding supernatants and incubated overnight at 4°C. Then Protein G plus/Protein A agarose suspension (Calbiochem, Switzerland) was added to the samples and incubated 2 hours at 4°C. Then the products were separated by 12% SDS-PAGE and NF-κB p65 was detected with its antibody (Cell signaling, Danvers, MA,USA).
Data are expressed as mean ±
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