Treatment of hypertension by increasing impaired endothelial TRPV4‐KCa2.3 interaction

Abstract The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta‐blockers, constipation by Ca2+ channel blockers, and dry cough by ACEI, lead to non‐compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca2+‐activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high‐salt diet, N(G)‐nitro‐l‐arginine intake, or angiotensin II delivery, showed decreased TRPV4‐KCa2.3 interaction in ECs. Perturbation of the TRPV4‐KCa2.3 interaction in mouse ECs by overexpressing full‐length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small‐molecule drug, JNc‐440, which showed affinity for both TRPV4 and KCa2.3. JNc‐440 significantly strengthened the TRPV4‐KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc‐440 specifically targeted the impaired TRPV4‐KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4‐KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.

Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the Reviewers whom we asked to evaluate your manuscript. I again apologise for the unusual delay in reaching a decision on your manuscript. In this case, we first experienced significant difficulties in securing expert and willing reviewers. I eventually only managed to secure two reviewers. Further to this, the evaluations were delivered with some delay.
I am therefore proceeding based on the two evaluations obtained so far as further delay cannot be justified and would not be productive.
As you will see, although the reviewers find your work potentially interesting and relevant, they both point to important technical and formal. For instance reviewer 2 notes two fundamental points, i.e. lack of direct experimental support that TRP4 and KCa2.3 interact and lack of understanding of how JNC-440 performs compared with other anti-hypertensives.
Our reviewer cross-commenting exercise confirmed the positive stance but also the need to address the issues as a requirement for publication.
In conclusion, while publication of the paper cannot be considered at this stage, given the potential interest of your findings and after internal discussion, we have decided to give you the opportunity to address the criticisms.
We are thus prepared to consider a substantially revised submission, with the understanding that the Reviewers' concerns must be addressed with additional experimental data where appropriate and as outlined above, and that acceptance of the manuscript will entail a second round of review. The overall aim is to significantly upgrade the relevance and conclusiveness of the dataset, which of course is of paramount importance for our title.
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Last, but not least, please carefully conform to our author guidelines (http://embomolmed.embopress.org/authorguide) to ensure rapid pre-acceptance processing in case of a favourable outcome on your revision. I look forward to seeing a revised form of your manuscript in due time. ***** Reviewer's comments ***** Referee #1 (Remarks): The authors addressed to the function of small arteries in terms of endothelia K+ channels and EDHF signaling to treat hypertension. They identified a reduced interaction between TRPV4 and KCa2.3 in EC from hypertensive patients and murine hypertensive model. They further developed a small-molecule drug, JNc-440, which strengthened the TRPV4 and KCa2.3 coupling in ECs and enhanced vasodilation and exerted antihypertensive effects in mice. These results are interesting to explore the novel antihypertensive drug. Our concerns are as follows.
1. In Figure 1g, ACh-induced vasodilation and sodium nitroprusside-induced vasodilation should be shown to clarify the EDHF or NO-dependent vasodilation. 2. In terms of the results from ACh-induced vasodilation in several experiments, the results are similar to that from GSX-induced vasodilation in hypertensive mice model, but not in TRP4-KO mice ( Figure 3C and 3f). How could you explain about the discrepancy? 3. Similarly, in Figure 4d, because L-NNA is known as an inhibitor for NO, the reviewer would like to know whether the effect of Ach is NO-independent or not. In Fig. S5c, the effect of caveolin-1 colocalization also affect Ach-dependent vasodilation. 4. In Figure 4e-g, the mean arterial blood pressure was shown in each group. Although the time course of this data is one week after venous injection, could you show the acute effect of JNc-440 in blood pressure? They demonstrated that the amount of TRPV4-KCa2.3 coupling is observed as early as 2 hours after venous injection. Thus, the time course of two experiment is quite different.

General Comments:
The authors addressed to the function of small arteries in terms of endothelia K+ channels and EDHF signaling to treat hypertension. They identified a reduced interaction between TRPV4 and KCa2.3 in EC from hypertensive patients and murine hypertensive model. They further developed a small-molecule drug, JNc-440, which strengthened the TRPV4 and KCa2.3 coupling in ECs and enhanced vasodilation and exerted antihypertensive effects in mice. These results are interesting to explore the novel antihypertensive drug. Our concerns are as follows. Answer: Thank you for your generous comments. Figure 1g, ACh-induced vasodilation and sodium nitroprusside-induced vasodilation should be shown to clarify the EDHF or NO-dependent vasodilation. Answer: Thank you for your comments. We have now followed your suggestion and performed the experiments.

Specific Comment #1) In
(1) In freshly isolated small arterial segments taken from mice that had been fed a high-salt diet, acetylcholine (ACh) induced relaxation was weakened, but sodium nitroprusside (SNP) relaxation was not significantly different (Figures for Referees not shown.). The results are consistent with previous reports 1 and suggesting the high-salt diet weakens EDHF-dependent, but not nitric oxide (NO)-dependent vasodilation.
(2) Consistently, membrane potentials were measured by sharp microelectrodes impaled from adventitial side in small arteries, we shown that the smooth muscle hyperpolarization, which is an indicator of EDHF release [2][3][4] , was activated by TRPV4 channel agonist GSK1016790A or ACh.  Figure 3C and 3f). How could you explain about the discrepancy? Answer: TRPV4 channels play a major role in endothelial-dependent vasodilation 5 . Either GSK1016790A or ACh could activate TRPV4 channel. The former is synthetic, but specific 6,7 ; the latter is natural, but not specific. It is reported that, in TRPV4 KO mice, GSK1016790A-induce response was completely lost 8 , but ACh-induced response was impaired about 20-50% 9, 10 . Our data is consistend with previous reports. This is due to the different specificity of GSK1016790A and ACh to TRPV4 channel. The before-mentioned information has been added into manuscript as discussion of fig. 2d and g (similar to fig. 3c compared to fig. 3f). Figure 4d, because L-NNA is known as an inhibitor for NO, the reviewer would like to know whether the effect of Ach is NO-independent or not. In Fig. S5c, the effect of caveolin-1 colocalization also affect Ach-dependent vasodilation. Answer: Thanks for your comments. We followed your suggestions and performed additional experiments.

Specific Comment #3) Similarly, in
(1) In isolated small artery segments from L-NNA induced hypertensive mice, pre-incubation with nitric oxide synthase inhibitor L-NAME did not abolish ACh-induced (Figures for Referees not shown.). This result indicated that the effect of ACh is NO-independent. Previous reports showed that L-NNA delivery is a common animal model for hypertension 11,12 . In this animal model, NO pathway was inhibited in vivo. Thus in small artery, compared to the role of NO, EDHF is dominant 2, 13 .
(2) The results in the previous version of manuscript, it is suggested that caveolin-1 colocalization also affect ACh-dependent vasodilation. In addition to pharmacological agent, we now further used AAV-siRNA-caveolin-1. We found that AAV-siRNA-caveolin-1, decreased TRPV4-KCa2. , which sustained as long as 8-12 h, with a dose of 1 mg/kg. We also recorded blood pressure in time-course in normotensive wild-type mice for 12 days (144 h) and no marked effect on blood pressure was observed (Fig. S10g). This result and results from fig. 4f together suggest antihypertensive effect of JNc-440 was absent in normotensive mice as long as 12 days of observation. These information has been added into the manuscript.
(2) In the previous version of manuscript, we demonstrated that in high-salt, L-NNA and AngIItreated mice, JNc-440 showed significant antihypertensive effects one week after tail injection (Fig.  4e) with a minimum dose of 1 mg/kg (Fig. S7b). We made confusion here about different time course. It should be JNc-440 (q.d.; one week) still showed significant antihypertensive effects after tail injection. We made this modification in the current manuscript. (2) Although WT and TRPV4 KO animals became hypertensive after each treatment, our results and previous studies showed that the resulting level of blood pressure was greater in TRPV4 KO mice compared with WT controls, but not significant 14 . We thought that in TRPV4 KO mice, there is a systematic change in these mice such as metabolic pathway and EC function, so that blood pressure regulation is complicated. During high salt, L-NNA and AngII treatment in TRPV4-KO mice, absence of TRPV4 may be compensated by other factors in circulation system to regulate blood pressure. In the further, we are planning to establish TRPV4/KCa2.3 knockout especial in EC, which could further explore the role of TRPV4/KCa2.3 in vivo. Further, enlightened by the reviewer's question, we have tried to identify these factors by using metabonomics technology. We have now finished the primary metabonomics analysis in high salt, L-NNA and AngII treatment in WT and TRPV4-KO mice, and identified several factors may be involved. We are glad to share our new results in future.

General Comments:
This work performed by Dongxu He and his colleagues uncovered a previous unrecognized connection between TRPV4 and KCa2.3, which was critical to the development of hypertension. Moreover, authors also provided an effective intervention drug Jnc-440, which augmented the TRPV4/KCa2.3 coupling to counteract high blood pressure without affecting their activities. This work was well-performed and written with novelty. However, some results are necessary be optimized to strengthen the conclusion. Here list some suggestions and comments. Answer: Thank you for your generous comments.

Specific Comment #1)
Most of the TRPV4-KCa2.3 coupling results were presented using immunofluorescent staining (FRET). Some other experiments detecting the interaction between the two molecules, such as co-IP, should be also used to further support the critical conclusion that this coupling was a critical step in the process of hypertension. Also, the effect of caveolae should be also confirmed. Answer: Thanks for your comments. We followed your suggestions and performed additional experiments. We used co-IP to confirm the interaction of caveloin-1/TRPV4/KCa2.3.

Specific Comment #2) The positive FRET signal depends on the physical colocalization of TRPV4 and KCa2.3. However, it is not an appropriate method to prove the existence of functional coupling between the two channels. Thus, additional experiments showing the functional interaction of TRPV4 and KCa2.3 should be included to validate this point.
Answer: In the previous version of manuscript, we measured intracellular K + and arterial tension. TRPV4 allows Ca 2+ influx, and KCa2.3 is Ca 2+ sensitive K + channel which allow K+ efflux. Thus, by activation of TRPV4 with specific TRPV4 agonist GSK1016790A, measurement of K + efflux and arterial tension could test the functional coupling. Following to the reviewer's suggestion, we further performed whole-cell patch clamp to test the functional coupling of TRPV4-SKCa3. In primarily cultured ECs, TRPV4 activator GSK1016790A induced a whole-cell current, which could be inhibited by TRPV4 inhibitor HC067047, but not by KCa2.3 inhibitor, apamin (Figures for Referees not shown.). In contrast, KCa2.3 activator, CyPPA, induced a whole-cell current, which could be inhibited by TRPV4 inhibitor, HC067047 (Figures for Referees not shown.). These results strongly supported that the functional coupling of TRPV4-KCa2.3 which also revealed by fluorescent K + efflux measurement in the previous version of manuscript.

Specific Comment #3)
The reference describing cyclodextrin needs be added. In addition, the effect of cyclodextrin should be confirmed by adding AAV-siRNA-caveolae in primary ECs. Answer: (1) the reference describing methyl β-cyclodextrin (MβCD) was added in the current version of manuscript 15,16 .
(2) In addition to pharmacological agent, we further used AAV-siRNA-caveolin-1. We found that AAV-siRNA-caveolin-1 decreased TRPV4-KCa2.3 coupling and GSK1016790A-induced K+ efflux in primary ECs, as well as GSK1016790A-or acetylcholine (ACh)-induced vasodilation in freshly isolated arterial segments (Figures for Referees not shown.). The results are consistent with our former data.

Specific Comment #4) This work indicates that JNc-440 significantly strengthens the destroyed endothelial TRPV4-KCa2.3 coupling in hypertensive models. However, the authors did not show the effect of JNc-440 on normal physiological condition to prove the safety and specificity of this new drug. To emphasize its superiority, it is suggested that the anti-hypertensive effect of JNc-440 should be compared with other widely used anti-hypertensive drugs such as calcium channel antagonists and angiotensin II receptor blockers in discussion.
Answer: Thanks for your comments. We followed your suggestions and performed additional experiments. (1) We have shown that JNc-440 has no effect on blood pressure injected at 1 mg/kg/day in normotensive mice after one week of treatment (Fig. 4f). Here, we further showed the short-term effect of JNc400 on blood in normotensive mice within 24 h of treatment. Data showed that JNc-440 did not markedly affect blood pressure with intravenously injection at 1 mg/kg (Figures for Referees not shown.). We also recorded blood pressure in time-course in normotensive wild-type mice for 12 days (144 h) and no marked effect on blood pressure was observed (Fig. S11d). These results and results from fig. 4f together suggest antihypertensive effect of JNc-440 was absent in normotensive mice as long as 12 days of observation. This information has been added into the manuscript.
(3) We compared JNc-440 with other anti-hypertensive drugs in this manuscript mainly focus on the side effects: In addition to reducing blood pressure, currently available antihypertensive agents have undesirable adverse effects, due to the systemic actions of the drugs by systemically altering targets activity (receptors/channel/enzyme). These effects, such as loss of potassium ions for diuretics, bronchospasm for beta-blockers, constipation for Ca 2+ channel blockers, and dry cough for ACEI, edema caused by unmatched circulation between arteries and veins during treatment of Ca 2+ channel blockers 17 , lead to no adherence with therapies. We explored new antihypertensive method specifically targeting sites of dysfunction. We developed a small-molecule drug, JNc-440, which showed affinity to both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 coupling in ECs and enhanced vasodilation and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the destroyed TRPV4-KCa2.3 coupling in ECs but did not systemically activate TRPV4 and KCa2.3.
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Referee #2 (Remarks):
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