A Systematic Review on Rho-Kinase as a Potential Therapeutic Target for the Treatment of Erectile Dysfunction

Figures & data

Table 1 Databases Employed and Respective Keywords Used

	Search Arm	Search Terms Used (Free Text or MeSH Terms)
#1	Rho-kinase	Rho-kinase OR Rho-kinase inhibitors OR ROCK
#2	Erectile dysfunction	Erectile dysfunction OR Sexual dysfunction

Notes: The search was made on PubMed, Scopus, Google Scholar and manual search. Search terms within the same arm were combined by “OR” and these search arm #1 and #2 were combined by “AND”.

Figure 1 Schematic representation of the data extraction process.

Table 2 The Quality Assessment of Individual Study Obtained According to Modified CAMARADES Checklist Items

References	I	II	III	IV	V	VI	VII	VIII	IX	X	Total (of 10)
^Citation33	1	0	1	1	1	0	1	1	1	1	8
^Citation17	1	0	1	1	0	0	1	1	1	1	7
^Citation34	1	0	1	1	0	0	0	1	1	1	6
^Citation35	1	1	1	1	0	0	1	1	0	1	7
^Citation36	1	1	0	1	0	0	1	1	0	1	6
^Citation37	1	1	0	1	0	0	1	1	1	1	7
^Citation41	1	1	1	1	0	0	1	1	1	1	8
^Citation19	1	1	0	1	0	0	0	1	1	1	6
^Citation44	1	1	1	1	1	0	1	1	0	1	8
^Citation40	1	1	1	1	1	0	1	1	1	1	9
^Citation38	1	1	1	1	1	0	1	1	1	1	9
^Citation42	1	1	1	1	1	0	1	1	1	1	9
^Citation43	1	1	0	1	1	0	1	1	1	1	8
^Citation20	1	1	1	1	1	0	1	1	1	1	9
^Citation45	1	0	1	1	0	0	1	1	1	1	6
^Citation46	1	0	1	1	0	0	1	1	0	1	6
^Citation47	1	1	1	1	1	0	1	1	1	1	9
^Citation21	1	0	0	1	0	0	1	1	0	1	5

Notes: I: Publication in a peer-reviewed journal, II: Number of experimental and control groups, III: Housing and Husbandry Conditions, IV: Details of intervention/exposure group procedures, V: Random allocation to groups, VI: Concealment of allocation, VII: Blinded assessment of outcomes, VIII: Biochemical evaluations, IX: Histopathological evaluations, X: Statistical analysis 1: Criterion is satisfied, 0: Insufficiently described or not explained at all; the mean quality score is expressed as mean ± standard error of the mean (SEM) and (minimum and maximum score).

Table 3 Role of Rho Kinases (ROCK) in the Pathogenesis of Erectile Dysfunction

Model and Method Used	Intervention and Process	Treatment Outcomes and Conclusion	References
Bilateral CN crush injury in 10-weeks old male Sprague Dawley (SD) rats.	Electro-stimulation was performed to assess erectile function. Penile tissue was processed for Masson’s trichrome staining, Western blot and double immunofluorescent staining.	A significantly lower percent of ratio of maximal ICP/MAP and areas under the ICP curve to MAP (AUC/MAP). A significantly higher expression of ROCK I/LIMK II in injured groups as compared with controls. Conclusion: The ROCK I/LIMK2/cofilin pathway may be involved in the ED.	^Citation33
Ligation of the left anterior descending coronary artery (HF) induced ED in rats	Electro-stimulation was performed to assess erectile function. RhoA, Rho-kinase II (ROCK II) and myosin phosphatase target protein 1 (MYPT-1) protein expression and phosphorylation levels were determined by Western blot analysis.	HF rats display impaired erectile function represented by decreased ICP/MAP responses. HF decreased RhoA protein expression, but increased ROCK II and MYPT-1 phosphorylation levels in cavernous tissue. Conclusion: Increased responses in cavernosal tissue, suggesting the involvement of ROCK signaling pathway in ED genesis.	^Citation17
Streptozotocin (STZ) induced DM on C57BL/6 and NOX1 knockout mice.	DM was induced by STZ. Functional properties of internal pudendal arteries (IPA) were assessed using a myograph, protein expression and peroxiredoxin oxidation by Western blot RNA expression by a polymerase chain reaction.	IPA from diabetic mice displayed increased contractions to phenylephrine. High glucose increased ROS generation in IPA vascular smooth muscle cell. High glucose increased levels of nitrotyrosine, protein oxidation/carbonylation, and Rho kinase activity Conclusion: Rho-kinase activation, via NOX1-derived ROS and downregulation of Nrf2 system, impairs IPA function in DM.	^Citation34
Age-associated ED was assessed in SD rats.	Erectile response measurements were performed by ICP/MAP ratio. Detection of Rho-kinase and eNOS protein was done by Western blot analysis. Finally, correlation analyses of the association between ICP/MAP and Rho-kinase, eNOS, or eNOS/Rho-kinase, as well as between age and eNOS or Rho-kinase, were performed.	The functional index ICP/MAP decreased with age in SD rats. The expression of eNOS protein decreased, while Rho-kinase expression increased. (eNOS/Rho-kinase) ratio decreased with age. eNOS was found to be significantly negatively correlated with age, whereas Rho-kinase was positively correlated with age. Conclusion: Age-associated ED was therefore correlated with decreased eNOS and increased Rho-kinase.	^Citation35
Vasculogenic and post-radical prostatectomy induced ED in human subjects	Samples were collected from individuals who undergo penile surgery for Peyronie’s disease and from men with ED who underwent penile prosthesis implantation. ED was categorized into vasculogenic and post-RP subtypes. Penile erectile tissue samples were collected for molecular analyses of protein expressions of nNOS and eNOS, (Ser-1412, (Ser-1177), (Ser-473), PDE5, a-smooth muscle actin phosphomyosin phosphatase target subunit 1, RhoA/ROCK-I/ROCK-II by Western blot.	Vasculogenic ED was characterized by decreased eNOS protein expression and eNOS and eNOS phosphorylation on their activatory sites (Ser-1177 and Ser-1412, respectively), uncoupled eNOS, upregulated PDE5 protein expression, increased ROCK activity, and increased oxidative stress in erectile tissue. Post-RP ED was characterized by decreased eNOS protein expression, increased eNOS phosphorylation on its activator site (Ser-1412), uncoupled eNOS, down-regulated PDE5 protein expression, and increased oxidative stress in erectile tissue.	^Citation36

Abbreviations: CN, cavernous nerve; DM, diabetes mellitus; ED, erectile dysfunction; eNOS, endothelial nitric-oxide synthase; ICP, intracavernosal pressure; MAP, mean arterial pressure; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; ROCK, Rho-associated coiled-coil-forming protein kinase.

Table 4 Role of Rho Kinases (ROCK) Inhibitors as a Novel Therapeutic Agent for the Treatment of Erectile Dysfunction

Model/Method	Intervention and Process	Treatment Outcomes and Conclusion	References
Relaxation of CC tissue on patients with ED after penile prosthesis implantation.	Human CC samples were obtained from individuals undergoing penile prosthesis implantation. qPCR was performed for the expression of RhoA and ROCK subtypes 1 and 2. Immunohistochemistry staining against ROCK and α smooth muscle actin (α-SMA) was performed on CC of an ED patient. Effect of Y-27,632 and vardenafil were assessed.	The expression of ROCK I was unchanged, while ROCK II was significantly upregulated in ED patients. After incubation with DMSO, Y-27,632 relaxed pre contracted tissues with 85.9 ± 10.3% (p = 0.0016 when compared to vehicle). Additive effects on the relaxation of human CC were seen after pre-incubation with 1μM vardenafil.	^Citation37
DM-induced ED (DMED) in rats	Type 1 DM was induced by STZ. Eight weeks later, the erectile function of rats was assessed with an Apomorphine test. Role of FTY720 was assessed Metabolic parameters; erectile function; sphingosine-1-phosphate receptor 3 (S1P3), protein kinase B (Akt), nitric oxide (NO), and cGMP signaling pathway; corporal fibrosis; apoptosis level; and Smad and non-Smad signaling pathways were assessed.	Erectile function in the DMED group was significantly impaired but significantly, improved in the DMED+ FTY720 group. The DMED group showed inhibited activity of the S1P3-Akt-NO-cGMP signalling pathway, and the inhibition was partly reversed in the DMED + FTY720 group. The DMED group showed serious corporal fibrosis, higher apoptosis level, a higher level of Rho-kinase, LIM domain kinase 2, and cofilin. FTY720 supplementation partly inhibited the activity of Rho-kinase, LIM domain kinase 2 (the Smad and non-Smad pathways).	^Citation38
DM-induced ED (DMED) in rats	After grouping DM was induced in rats via STZ Eight weeks after DM induction, rats with ED were selected via an Apomorphine test. JTE-013 was administered intraperitoneally for treatment groups. The rest were fed under the same condition. Erectile function was measured by CN electro stimulation. The expression levels of related signaling pathways were evaluated using Western blotting, real-time PCR and immunohistochemistry.	Erectile function was significantly impaired in the DMED group and was partially improved in the DMED + JTE-013 group. The expression RhoA/ROCK/p-MYPT1 pathway proteins were higher in the DMED group and JTE-013 treatment significantly reduced the expression/activity of these proteins. Conclusion: JTE-013 supplementation significantly ameliorated these pathological changes	^Citation19
Clinical and DM-induced ED (DMED) in rats	DM was induced by administering STZ. Apomorphine was used to assess erection. Role of microRNA-141 was assessed Hematoxylin-eosin (HE) staining and Masson staining, Immunohistochemistry, Quantitative real-time polymerase chain reaction qRT-PCR, cell culture and western bloating	The mRNA and protein expressions of RhoA and ROCK II were significantly increased while the expression of microRNA-141 was decreased in the penile tissues. The microRNA-141 expression in the microRNA-141 inhibitors + siRNA-Rho group was significantly decreased. MicroRNA-141 specifically bound to Rho-3ʹ-UTR and down-regulated the expression of the Rho gene at the post-transcriptional level.	^Citation20
Partial bladder outlet obstruction (PBOO) in a rat model.	After animals were grouped, PBOO was induced by ligation of the urethra for 6 weeks. In vivo, erectile responses were monitored by evaluating ratios of ICP/MAP ratio. Organ-bath studies were performed on CC strips. Penises were assessed at baseline for protein expression of eNOS and ROCK-II by Western blot.	The ratio of ICP/MAP was significantly decreased in obstructed rats, which was restored after treatment eNOS expression in the obstructed group decreased, which was improved by treatment. However, there was no significant difference in the protein levels of ROCK II between groups.	^Citation40
Age-associated corporal fibrosis in SD rats.	Effect of human tissue kallikrein 1 (hKLK1) on TGR harboring the hKLK1 gene was fed to 4- or 18-month-old rats and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). The erectile function of all rats was assessed by CN electrostimulation method. Masson’s trichrome staining and Western blotting were used to evaluate corporal fibrosis in the CC.	The erectile function of rats in the aWTR group was significantly lower than the other two groups. Immunohistochemistry and Western blotting showed that expression of transforming growth factor-β 1 (TGF-β1), RhoA, ROCK I, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group and improved in hKLK1 treated groups. Conclusion: hKLK1 may reduce this corporal fibrosis by inhibiting the activation of Rho-kinase.	^Citation41
CN-crush injury in male SD rats.	Animals were grouped into 3 groups, including sham surgery, CN crush injury and CN- crush injury treated with fasudil. Effect of Fasudil was assessed Electro stimulation and dynamic infusion cavernosometry were performed postoperatively. Penile tissue was processed for immunohistochemistry, double immune fluorescent and Masson trichrome staining, TUNEL, caspase-3 activity assay and Western blot.	The CN crush injury group showed significantly lower ICP/MAP. Rho-kinase inhibition in the injury plus fasudil group restored erectile responses. Conclusion: Rho-kinase inhibition in the injury plus fasudil group alleviated the histological and molecular dysregulation.	^Citation42
Methylglyoxal (MGO) administered male rats.	The effect of exendin-4 (Ex-4) treatment on CC dysfunction was assessed. Animals were grouped into four groups as control, MGO, MGO + low-dose Ex-4 and MGO + high-dose Ex-4. Endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), NADPH oxidase subunit gp91phox (NOX2), and Rho kinase (ROCK II) expressions in CC were investigated by immunohistochemistry.	In MGO administered rats, both endothelium-dependent and neurogenic CC relaxations were significantly impaired. The diminished response was significantly improved by exendin-4 treatment. Conclusion: Exendin-4 treatment improves NO-mediated CC relaxations in MGO administered rats probably by inhibiting NADPH oxidase.	^Citation43
DM induced ED in a rat model.	Role of angiotensin (Ang) II inhibitor (short hairpin RNA (shRNA)) was assessed. ICP/MAP was measured after electrical stimulation. Western blotting and quantitative RT-PCR were applied to measure the expressions of RhoA, ROCK-I and II. Radioimmunoassay was applied to detect the levels of Ang II.	Rats with DMED had worse ICP and MAP than Ang-II-silenced rats. Prolonged erectile time had shown in Ang-II-silenced rats. The contraction ability was markedly improved and relaxation ability was decreased in Ang-II-silenced rats. The mRNA and proteins of RhoA and ROCK II were expressed in a similar way. Conclusion: Ang-II silencing improves ED via down-regulating the RhoA/Rho-kinase signaling pathway.	^Citation44
Patients who had overactive bladder (OAB) and ED and Mirabegron induced CC relaxation in animal models	CC specimens were obtained from patients with ED and Peyronie’s disease undergoing penile prosthesis implantation. Erectile responses were evaluated in vivo after intracavernosal injection (ICI) of mirabegron in anaesthetized rats. Mirabegron-elicited relaxation responses on phenylephrine-induced contraction were seen in human CC (HCC) and rat CC strips in isolated organ bath studies. The effects of inhibitors, namely L-NAME, ODQ, methylene blue, SR59230A and fasudil on mirabegron-induced relaxation, responses were evaluated. Immunohistochemistry was used to localize b3-adrenoceptors and ROCK in CC smooth muscle cells.	Mirabegron resulted in a relaxation of phenylephrine evoked CC and SR59230A antagonized the mirabegron induced relaxations in HCC and rat CC. Mirabegron relaxation was enhanced by fasudil (ROCK inhibitor) in rat but not in HCC strips. Immunohistochemistry data showed b3-adrenoceptors localized in the smooth muscle cells of the HCC and rat CC.	^Citation45

Abbreviations: CN, cavernous nerve; CC, corpus cavernosum; DM, diabetes mellitus; ED, erectile dysfunction; eNOS, endothelial nitric-oxide synthase; ICP, intracavernosal pressure; MAP, mean arterial pressure; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; ROCK, Rho-associated coiled-coil-forming protein kinase; STZ, streptozotocin.

Table 5 Traditional Plants Used as Rho Kinases (ROCK) Inhibitors as a Therapeutic Agent for the Treatment of Erectile Dysfunction

Model/Method	Intervention and Process	Treatment Outcomes/Conclusion	References
The ex-vivo activity was done on the isolated CC of rabbits.	Isolated rabbit CC strips were mounted in an organ bath system. Effect of Eupatilin (the main compound present in Artemisia species) A conventional whole-cell patch-clamp technique was used to measure activation of calcium-sensitive K+-channel currents in human corpus cavernously smooth muscle (CCSM) cells. The relaxation effect of Eupatilin was evaluated by cumulative addition to CC strips precontracted with phenylephrine. Western blotting analysis was performed to measure MYPT1 and protein kinase C and to evaluate the effect of Eupatilin on the RhoA/Rho-kinase pathway.	Eupatilin effectively relaxed the phenylephrine-induced tone in the rabbit CC. Eupatilin reduced the phosphorylation level of MYPT1 at Thr853 of MLCP and CPI-17 at Thr38. Eupatilin-induced relaxation of the CCSM cells via NO-independent pathways. Conclusion: The relaxation effects of eupatilin on CCSM cells were partially due to activation of Ca^+2 activated K^+ (BKCa+2) channels and inhibition of RhoA/Rho-kinase	^Citation46
Bilateral CN injury (BCNI) induced ED in a rat model.	Effect of HongJing I (HJI) was assessed Rats were divided into five groups: normal control (NC), BCNI-induced ED model (M), M + low-dose HJI (HL), M + medium-dose HJI (HM), and M + high-dose HJI (HH). All groups were treated with normal saline or the relevant drug for 28 days after inducing BCNI-ED. Finally, ICP was recorded, Immunofluorescence staining and Western blotting were applied to detect the changes in fibrosis protein and RhoA, ROCK I, and ROCK II expression.	In addition, RhoA, ROCK1, and ROCK2 expression levels were increased upon BCNI-ED induction. HJI effectively improved the ICP in the treatment groups. HJI successfully inhibited cavernosum fibrosis and the activation of RhoA/ROCK2 signaling. Overall, these results suggest that the effects of HJI in attenuating ED may be caused, at least in part, by the suppression of RhoA/ROCK II signaling and alleviation of fibrosis.	^Citation47
In vitro, ROCK II Kinase assay was done.	Effect of Eurycoma longifolia (EL) Jack and Y- 27,632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4 pyridyl) cyclohexane carboxamide dihydrochloride] was used as standard drug. Luminescence was recorded using Topotecan, USA, Spark 10 M, multimode microplate reader. Standard curve for ROCK-II enzyme was done. Serial dilution and IC 50 of the AE was performed in triplicate	EL and Y-27,632 as a standard showed a significant inhibition for ROCK-II activity. The inhibition of ROCK-II activity at P < 0.001. IC 50 in ROCK-II inhibition assay of EL (651.1 ± 32.9 ng/mL). The traditional use of E. longifolia as aphrodisiac and for male sexual disorders might be partially due to the ROCK-II inhibitory activity.	^Citation21

Abbreviations: CCSM, corpus cavernously smooth muscle; CN, cavernous nerve; DM, diabetes mellitus; ED, erectile dysfunction; ICP, intracavernosal pressure; MAP, mean arterial pressure; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; ROCK, Rho-associated coiled-coil-forming protein kinase.

Figure 2 RhoA/Rho-kinase signaling pathway for Ca²⁺ sensitization in cavernosal smooth-muscle cells.

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