TRPV4 regulates migration and tube formation of human retinal capillary endothelial cells

Background Ca2+ entry plays an important role in modulating endothelial cell migration and tube formation. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a Ca2+-permeable channel that is widely expressed in endothelial cells. It has been reported that TRPV4 is expressed in HRCECs and regulates Ca2+ entry. However, the function of TRPV4 in human retinal capillary endothelial cells (HRCECs) remains unknown. Methods In this study we used western blot and immunostaining assay to verify TRPV4 expression in HRCECs. And then we pretreated HRCECs with HC067047 and transfected with specific shRNA of TRPV4. The functional presence of TrpV4 was determined by using fluorescence, migration and tube formation assay in TrpV4 knockdown cells or control cells. Results Using western blot and immunostaining, we confirmed TRPV4 expression in HRCECs. Moreover, inhibition of TRPV4 using the specific inhibitor HC067047 and the knockdown of TRPV4 with shRNA significantly suppressed tube formation and migration by HRCECs. Conclusions TRPV4 is essential for HRCEC migration and tube formation, and maybe a potential therapeutic target for retinal vascular diseases. Electronic supplementary material The online version of this article (10.1186/s12886-018-0697-2) contains supplementary material, which is available to authorized users.

Recently, several studies have focused on the function of TRPV4 in the retinal microvascular endothelium of rats and mice [9]. However, human-derived ECs have not been studied. During retinal development, the activation and migration of ECs are important in forming tubular structures. However, retinal neovascularization is also a characteristic pathological consequence of many retinal diseases, including retinopathy of prematurity [10], diabetic retinopathy [11], and retinal vein occlusion [12].
In previous studies, we showed that TRPV4 regulates flow-induced endothelial Ca 2+ entry [13] and vascular function [14]. Besides, the TRPV4-KCa2.3 signaling pathway plays an important role in smooth muscle hyperpolarization and relaxation and may be involved in endothelium-derived hyperpolarizing factor dysfunction in diabetic rats [15]. Here, we show that TRPV4 is expressed in human retinal capillary ECs (HRCECs) and is essential for their migration and tube formation.

Immunostaining assay
HRCECs were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100(Invitrogen, Grand Island, NY, USA) for 10 min at room temperature. After blocking with 5% bovine serum albumin in PBS for at least 30 min at room temperature [18], the cells were incubated with specific primary antibodies against TRPV4 overnight at 4°C, and then with Alexa Fluor secondary antibodies(Invitrogen, Grand Island, NY, USA) for 2 h at room temperature. Cells were washed five times in PBS after each step. The images were captured using a confocal microscope.

Migration assay
Transwell chambers with a 0.4-μm pore size membrane. Cells were seeded at 3.8 × 10 4 in 100 μl serum-free DMEM in the top of a transwell chamber (353,097, corning, NY, USA) [20]. DMEM with 10% FBS (100 μl) was added to the lower chamber. After 48 h incubation at 37°C in a humidified atmosphere of 5% CO 2 , cells migrated to the lower surface of the chambers. And then, the chambers were removed and washed twice in PBS. The cells were fixed in 4% paraformaldehyde for 30 min and stained with crystal violet. Non-migrating cells were removed from the upper chamber with cotton-tipped swabs. Images were captured with a video camera (Coolpix 54, Nikon, Japan).
Tube-formation assay 50 μl Matrigel (354,234, BD Bioscience) was added to each well of a 96-well plate and incubated for 30 min at 37°C in a humidified atmosphere of 5% CO 2 [19]. Until matrigel was polymerized, Cells (4 × 10 4 in 100 μl DMEM) were seeded in each well. Capillary-like tubes were formed within 8 h and recorded with the video camera (Coolpix 54, Nikon, Japan).

TRPV4 expression in HRCECs
To confirm that TRPV4 is expressed in HRCECs, we performed western blots (Fig. 1a) and immunostaining assays (Fig. 1b) [22] on cell lysates of HEK293 cells and HRCECs. All the data showed that HRCECs express TRPV4.

TRPV4 regulates Ca 2+ entry into HRCECs
Our previous experiments had demonstrated that TRPV4 is expressed in HRCECs, so we then investigated its function using Ca 2+ measurements. We found a robust increase in intracellular Ca 2+ levels after stimulating cells with 100 nM GSK (a specific TRPV4 agonist) (Fig. 2a, c), while pretreatment with HC067047 (a specific TRPV4 antagonist) [23] significantly inhibited this effect (Fig. 2b, c). These data showed that TRPV4 is important in GSK-induced Ca 2+ influx into HRCECs.

Effect of TRPV4 on HRCEC migration
To evaluate the role of TRPV4 in HRCEC migration, we performed transwell assays with or without a TRPV4 inhibitor. The results showed increased Ca 2+ influx in GSK-stimulated HRCECs (Fig. 3). However, this effect was blocked when HRCECs were pretreated with the specific TRPV4 inhibitor HC067047 and the specific shRNA. These results demonstrated that TRPV4 plays a key role in the migration of HRCECs.

Effect of TRPV4 on HRCEC tube formation
In addition to migration, we studied the role of TRPV4 in tube formation by HRCECs. As expected, GSK increased tube formation (Fig. 4b), while pretreatment with HC067047 and shRNA blocked the effect (Fig. 4c, d). The results showed that almost no tubular structures formed after co-incubation of HRCECs with HC067047 and GSK, and also we verified the same results with HRCECs, which were transfected with specific shRNA of TRPV4. (Fig. 4f, g).
All these results suggested that TRPV4 is involved in tube formation by HRCECs.

Discussion
In this study, we assessed the role of TRPV4 in regulating HRCEC migration and tube formation. Using western blotting and immunostaining, we first confirmed that TRPV4 is expressed in HRCEC. The results of Ca 2+ imaging demonstrated that TRPV4 regulated Ca 2+ influx in HRCECs. By using the specific TRPV4 inhibitor HC067047 and using shRNA, we showed that inhibition or knockdown of TRPV4 significantly suppressed migration and tube formation by HRCECs.
Recently, several studies have demonstrated that TRP channels play vital roles in retinal epithelial, corneal, and endothelial cells. For instance, the decreased level of TRPV4 induced by hyperglycemia and diabetes contributes to diabetes-induced endothelial dysfunction and retinopathy in retinal microvascular endothelium [9]. Thermo-sensitive TRPV4 activation protects human corneal ECs [24]. Activation of TRPV4 modulates the extracellular melatonin in human non-pigmented ciliary ECs [25]. Activation of TRPV4 is necessary for the correct establishment of tight junctions in corneal epithelia as well as the regulation of both the barrier function of b a tight junctions and their ability to respond to epidermal growth factor [26]. However, no studies had focused on TRPV4 function in HRCECs. This is the first study, to our knowledge, to examine the role of TRPV4 in regulating HRCEC function. It is well known that TRPV4-regulated Ca 2+ influx is important for endothelial migration and tube formation, which are vital components of angiogenesis [27]. Also, dysfunctional angiogenesis may lead to several retinal diseases [1,28]. For example, functional expression of TRPV4 in retinal microvascular ECs are reduced in hyperglycemia and diabetes [9]; activation of TRPV4 in retinal ganglion cells leads to glaucoma and retinal detachment [29]; and TRPV4 is a target for treating ocular hypertension and conventional outflow by regulating Ca 2+ homeostasis and cytoskeletal remodeling [30]. These studies inspired us to test the hypothesis that TRPV4 is associated with human retinal diseases and can it be a potential therapeutic target.

Conclusions
In summary, we have provided direct evidence of functional TRPV4 expression in HRCECs and demonstrated that TRPV4 is involved in migration and tube formation in HRCECs.