High Levels of Heat Shock Protein 47 in the Aqueous Humor of Patients with Acute Primary Angle Closure

Primary angle-closure glaucoma has been identified as one of the primary types of glaucoma among Asians, especially in individuals of Chinese descent [1]. Sudden angle closure can result in rapid elevation of intraocular pressure (IOP) accompanied by severe ocular pain, headache, blurred vision, nausea, and vomiting. For patients with glaucoma whose symptoms do not improve with medications, trabeculectomy is regarded as one of the best methods to lower IOP especially in developing countries [2]. However, conjunctival bleb scarring has remained to be the most common cause for surgical failure, and the exact molecular mechanisms for scarring are yet to be determined.

Recent studies indicated that excessive collagen accumulation is one of the main pathological processes of bleb scarring [3, 4]. In our previous study, we demonstrated that upregulated expression of heat shock protein 47 (HSP47) correlated with increased postoperative deposition of types I and III collagen in rat conjunctival blebs and suggested that HSP47 may play a role in the progression of conjunctival bleb scarring [5]. Recent evidence demonstrated that HSP47 may be a candidate therapeutic target against fibrotic diseases and breast cancer [6‒9]. However, existing literature contains no information regarding HSP47 expression in human aqueous humor. To address this issue, we designed this study to explore the levels of HSP47 in the aqueous humor of patients with acute primary angle closure (APAC) and age-related controls.

This study was approved by the Ethics Committee of Xi’an Jiaotong University (No. 2015-107) and each institution according to the guidelines of the Declaration of Helsinki. Participants were recruited prospectively and consecutively at the Department of Ophthalmology in Xi’an People’s Hospital (Xi’an Fourth Hospital) from January 2021 to October 2021. Written informed consent was obtained from all participants.

The inclusion criterion was fulfilling the diagnostic criteria of APAC [10]. Meanwhile, the exclusion criteria were as follows: (1) secondary angle closure, (2) history of eye surgery, and (3) history of other eye diseases except for refractive error and cataract. Age-matched and -related patients with cataracts without glaucoma were enrolled as the control group and underwent routine phacoemulsification and intraocular lens implantation. All eyes of the participants underwent thorough ophthalmological evaluation, including slit-lamp biomicroscopy, IOP measurement, gonioscopy, funduscopic examination, UBM, and ocular B-scanning.

Patients with APAC were initially treated with the following topical and systemic medications to lower the IOP: 1% topical pilocarpine (Bausch and Lomb, Bridgewater, NJ, USA) four times daily; 2% topical carteolol (China Otsuka Pharmaceutical Co., Ltd. Shanghai, China) twice daily and/or brinzolamide (Alcon, Hunenberg, Switzerland), and/or brimonidine tartrate (Allergan, Dublin, Ireland) three times daily; oral methazolamide (Hangzhou AoyiPollenin Pharmaceutical Co., Ltd., Hangzhou, China) at 25 mg twice daily; and intravenous 20% mannitol of 250 mL (China Otsuka Pharmaceutical Co., Ltd.).

At the beginning of the surgery, 100–120 μL of aqueous humor was aspirated by limbal paracentesis. All samples were immediately stored at −80°C until analysis. The level of HSP47 within aqueous humor was measured using a commercial immunoassay kit (Heat Shock Protein 47 Human ELISA Kit, Abcam Inc.). All samples were diluted 1:1 for processing. A standard protocol was performed according to the manufacturer’s instructions.

Statistical Package for the Social Sciences (SPSS) version 22 (IBM Corp., Armonk, NY, USA) was utilized to analyze the data. Continuous data are displayed as means ± standard deviation, while categorical data are displayed as n (%). The two-tailed t test or χ² test was used for comparison of the data between the APAC and control groups. Correlations between HSP47 levels and subjects’ age, IOP, and time interval (between the onset of acute attack and sample collection) were calculated using Spearman’s correlation test. p < 0.05 was considered statistically significant.

In total, 32 patients with APAC were enrolled. The cohort consisted of 27 (84.4%) females, the mean age of the patients was 62.1 ± 8.0 years, and 19 (59.4%) left eyes were affected. The mean interval between the onset of acute attack and collection of aqueous humor samples was 6.8 ± 5.4 days. The characteristics of the 32 patients with APAC who fulfilled the inclusion criteria and 16 matched controls are summarized in Table 1.

The levels of HSP47 in the aqueous humor are shown in Table 2. In patients with APAC, the levels of HSP47 can be noted to be significantly higher than those in controls (p = 0.001). Figure 1 shows the distribution of HSP47 expression in the aqueous humor of individual patients, and Figure 2 shows the different age groups of both controls and patients with APAC along with the respective HSP47 levels.

The correlations between HSP47 and age, IOP1, IOP2, and interval in patients with APAC are shown in Table 3. The levels of HSP47 negatively correlated with the age of patients with APAC (p = 0.023), as shown in Figure 3.

HSP47, which is a collagen-binding stress protein, acts as a collagen-specific molecular chaperone during the biosynthesis and secretion of procollagen and is closely related to the pathogenesis of various fibrotic disorders, such as those in the lungs [11, 12], liver [13, 14], and cicatricial pemphigoid [15]. Hence, HSP47 can be considered a potential therapeutic target for fibrotic diseases and breast cancer [6‒9]. Our previous studies showed that the expression of HSP47 mRNA and protein in conjunctival blebs increased after filtration surgery in rats, and upregulated HSP47 correlated with the increased expression of types I and III collagen in conjunctival blebs after trabeculectomy [5]. Therefore, we proposed that HSP47 may play a role in conjunctival bleb scarring. However, whether HSP47 exists in human aqueous humor and how it influences conjunctival bleb scarring after trabeculectomy is yet to be determined.

In this present study, HSP47 was present in the aqueous humor of both patients with and without APAC, and the levels of HSP47 in patients with APAC were significantly higher than in those without APAC. This is one of the earliest studies to report the detection of HSP47 in the aqueous humor of patients with APAC using immunoassay.

Several studies have revealed possible molecular interactions between known fibrotic factors and HSP47. Razzaque et al. [15] disclosed that increased levels of HSP47 were detected in TGF-β1-treated conjunctival fibroblasts. Additionally, IL-4, IL-13, and connective tissue growth factor can increase levels of HSP47 in fibroblasts [16]. Furthermore, HSP47 affects collagen metabolism in scleral fibroblasts by promoting the synthesis and secretion of collagen I as well as inhibiting degradation [17]. As per Yamane et al. [18], it was found that increased levels of HSP47 in lacrimal gland fibroblasts may induce excessive formation of extracellular matrix, which could lead to chronic graft-versus-host disease fibrosis. Ohigashi et al. [19] explored vitamin A-coupled liposomes containing HSP47 small interfering RNA, which interferes with HSP47, to treat dry eye syndrome of ocular chronic graft-versus-host disease in a mouse model.

In an acute attack of glaucoma, levels of several cytokines in the aqueous humor were noted to increase [20‒23]. To the best of our knowledge, this present study is also the first to demonstrate the expression of HSP47 in the aqueous humor. We hypothesized that the increased levels of HSP47 in the aqueous humor of patients with APAC were produced by the anterior ocular cells in response to IOP elevation and that elevated levels of HSP47 in the aqueous humor may affect scleral or/and conjunctival fibroblasts, leading to scarring during the healing process after trabeculectomy. However, these hypotheses need to be further investigated.

The IOP measured on the first day of the patients’ hospitalization or on the day of surgery was not significantly correlated with the levels of HSP47. The possible reasons may be as follows: (1) IOP was reduced by antiglaucoma drugs, which may affect the aqueous microenvironment and influence the expression of HSP47; and (2) high levels of cytokines with upregulated expressions, which may interact with each other, were detected in patients with APAC. However, the mechanism of interactions among cytokines in the aqueous humor to modulate the IOP remains to be unclear. Further studies will be necessary to resolve these issues.

Another finding was that age was negatively correlated with levels of HSP47. This finding might demonstrate that HSP47 attenuates with increasing age, which is consistent with the study of Miyaishi et al. [24]. They found that there is age-related attenuation of the HSP47 heat response in fibroblasts. A novel technology to overcome the aging process is radiofrequency, which is known to generate heat and increase collagen formation via HSP47 induction [25]. Notably, the negative correlation of HSP47 with age may provide another possible explanation for the higher success rate of trabeculectomy in older patients with APAC.

Some limitations in our study should be mentioned. First, IOP-lowering medications may affect aqueous homeostasis. Second, the number of participants in this present study was relatively low, and the results and conclusions of this study should be strengthened by larger populations.

In conclusion, HSP47 was upregulated in the aqueous humor of patients with APAC compared with patients with cataracts without APAC. These findings may support the hypothesis that HSP47 may play a role in the pathogenesis of scarring after trabeculectomy in patients with APAC; however, the exact mechanisms need further investigation.

This study was approved by the Ethics Committee of Xi’an Jiaotong University (No. 2015-107) and each institution according to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants.

The authors have no conflicts of interest to declare.

This study was supported by the National Natural Science Foundation of China (No. 81500719) and Research Incubation of Xi’an People’s Hospital (Xi’an Fourth Hospital) (No.BS-2).

Wei-wei Wang designed the study. Hai-zhen Yu conducted the study. Xin Yang, Qian-qian Xu, and Huan-huan Yan collected the data; Xin Yang and Qian-qian Xu conducted statistical analysis; Jian-rong Liu participated in the study design. Wei-wei Wang drafted and revised the manuscript. All the authors have read and approved the final manuscript.

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.