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 
. Such an in vitro system provides precise control of the physicochemical environment for the investigation of molecular alternations in the mechanotransduction system, and allows precise measurements of consequent cellular functions. Pathological cyclic strain (15%) directly increased Rab28 expression in VSMCs, but not in ECs. Rab28 expression in ECs, however, increased significantly by the addition of culture CM from VSMCs subjected to the pathological cyclic strain. Combined with the effects of Rab28 siRNA transfection on EC functions, our results indicate that Rab28 expressed in ECs is susceptible to paracrine control by VSMCs and subsequently regulate EC homeostasis. We then investigated what is the bioactive substance in the CM from VSMCs that mediate the paracrine control of ECs.
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 
. Our present results revealed that Ang II concentration in the CM from VSMCs subjected to pathological cyclic strain is higher than that from VSMCs subjected to physiological cyclic strain. Furthermore, AT1R blockers attenuate the effect of CM from the strained VSMCs on the static ECs. These results indicate that VSMCs subjected to pathological cyclic strain might release Ang II to modulate Rab28 expression in ECs via a paracrine effect, which consequently affect EC functions.
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 
and the activated NF-κB then translocates into the nucleus to bind to chromosome to induce gene transcription 
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 
. Activation of NF-κB in arteries subjected to high intraluminal pressure prevents apoptosis in vascular cells 
. Our findings demonstrate that the Ang II synthesized by VSMCs subjected to high cyclic strain mediates the increase of Rab28 expression and the NF-κB activation in ECs. Rab28 increases the activation of NF-κB and then translocates with it into the nucleus. The activation of NF-κB eventually modulates gene transcription to modulate EC proliferation, apoptosis and migration ().
Schematic drawing outlines the possible role of Rab28 in EC homeostasis.
Large molecules (>40 kD) cannot pass through nuclear envelope freely. Their nuclear transport is controlled by the nuclear pore complex (NPC) 
. Proteins need karyopherins, including importins and exportins, to enter or leave the nucleus, respectively 
. Importins require nuclear localization signals (NLS) to identify which proteins could enter into the nucleus, while exportins search for nuclear export signals (NES) to identify which proteins could leave from the nucleus. As a result, karyopherin-cargo protein complex can pass through NPC. The separation of importin-cargo protein complex or combination of exportin with a cargo protein requires Ran GTPase 
, a nuclear Ras, to change the conformation of importin or exportin. Our present results suggested that, in addition to Ran, Rab28 might also regulate nuclear transport.
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.