Increase of Claudin-5, ICAM-1 and eNOS expressions in human brain endothelial cells by ammonium chloride

Introduction

Lysosomes are cytosolic organelles that play an important function in degradation mechanisms. It has been recognized to degrade unfolded proteins, aggregated proteins, superfluous proteins, damaged organelles, nucleic acids and toxic components [1, 2]. The degradative actions of lysosomes are mediated by its enzymes such as proteases, lipases, nucleases, phosphatases and other hydrolytic enzymes that break down those components for immediate processing [3]. It exerts maximal enzymatic activity at low pH (≤5). These lysosomal functions are part of autophagy process [4]. Autophagy is a crucial process for the conservation of the overall cellular homeostasis and prevent accumulation of those components in the cell [5]. Such functions are critical to provide protection against aging, cancer, neurodegenerative diseases, and infections [6].

Toxic proteins may accumulate in the cell if the degradation mechanisms could not encounter the overburden of protein misfolding or damaged organelles in the cell. Thus, defective or ineffective cellular clearance of long-lived cells and proteins could lead to the deposition of protein aggregates and exhibit increased evidence in the pathologies of neurodegenerative diseases such as Alzheimer’s, Parkinson’s and amyotrophic lateral sclerosis [7, 8]. Among common risk factors associated with these development are aging and oxidative stress, which lead to the etiology of neurodegenerative diseases [9].

Therefore, lysosome dysfunctions could affect cellular processes, since deficits in protein clearance can impair the balance, especially in the brain. The human brain endothelial cells (HBECs) have a pivotal role as part of the blood–brain barrier (BBB). These cells maintain the homeostatic mechanism by controlling the entry and exit of substances from the blood into the brain. Endothelial cells lining the cerebral blood vessels are the main structure that forming BBB. The tight junction (TJ) proteins glues endothelial cells to each other. Alterations or loss of TJ proteins such as claudin, could result in a compromised BBB [10]. Claudin-5 is highly expressed TJ protein in HBECs and its dysfunction has been demonstrated in neurodegenerative disorders, such as Alzheimer’s disease, multiple sclerosis and psychiatric disorders [11].

The intracellular adhesion molecule 1 (ICAM-1) is expressed on endothelial cells especially due to stimulation of proinflammatory cytokines and invokes a range of proinflammatory responses [12]. An increase in ICAM-1 enables the site for leukocyte binding to inflamed tissues during inflammation and could explain the state of cells in certain conditions [13]. Another important function of endothelial cells is property of vasodilation. The vascular tone is controlled by nitric oxide (NO) produced by NO synthase (NOS). The endothelial NOS (eNOS) controls vascular tone in order to change blood pressure and blood flow conditions under homeostasis and has been used as a marker in vascular dysfunctions [14].

Lysosomal activity impairment can contribute to the progression and detrimental effect to neurological conditions such as Alzheimer’s disease [15]. Lysosome dysfunctions could be induced by chemicals such as ammonium chloride. Ammonium chloride is a well known lysosomal function inhibitor. It has the ability increase the pH in lysosomes and inhibit fusion between autophagosomes and lysosomes autophagy process. Lysosome is an important component for degrading organelles and protein degradation and its dysfunction has been associated with neurodegenerative diseases such as Alzheimer. Reduced autophagy function leads to the accumulation of proteins, lysosomal vesicles contain toxic hydrolases, inflammatory cytokines, reduced eNOS, and increased ROS. A clear fundamental link need to be established on how impairment from the cellular level could affect cerebrovascular pathogenesis. Thus, this study intends to investigate whether lysosomal dysfunction could cause endothelial changes in terms of inflammation status (ICAM-1), vasodilation (eNOS) and tight junction protein expression (Claudin-5) that serve as the main molecules in maintaining physiological endothelial function. The in vitro model used in the present study may serve as a preliminary finding on the cellular changes caused by a lysosome inhibitor on HBECs.

Materials and methods

Results

Claudin-5 expression on human brain endothelial cells in control and treated samples

Figure 1 shows that the Claudin-5 protein expression was significantly increased (p<0.05) in cells treated for 1 h with 10 mM ammonium chloride. Control samples (1C) showed a protein expression of 20,313.6±1,637.1 pixels, whereas the treated group (1T) demonstrated a higher protein expression of 66,829.5±3,937.7 pixels. Based on the densitometry analysis, a higher intensity band of phospho-Claudin-5 was observed in the treated samples. Moreover, a higher chemiluminescence signal of phospho-Claudin-5 was detected in the treated samples compared to the control samples.

Figure 1:

Claudin-5 protein identification by capillary immunoassay. (A) Densitometry analysis and chemiluminescence signals of the control, (B) densitometry analysis and chemiluminescence signals of samples treated for 1 h with 10 mM ammonium chloride, (C) * indicates significant increase (p<0.05) of Claudin-5 expression in cells treated for 1 h with 10 mM ammonium chloride. Data are expressed in means±SEM. All experiments were conducted in triplicate.

ICAM-1 expression on human brain endothelial cells in control and treated samples

ICAM-1 protein expression was significantly increased (p<0.05) in the cells treated with 10 mM ammonium chloride (Figure 2). Control samples (1C), showed a protein expression of 2,592,090.0±159,696.0 pixels, whereas the treated group demonstrated a higher protein expression of 515,588.7±448,555.0 pixels. Based on the densitometry analysis, a higher intensity band of ICAM-1 was observed for the treated samples compared to the control samples. Moreover, a higher chemiluminescence signal of ICAM-1 was detected in the treated samples compared to the control samples.

Figure 2:

ICAM-1 protein identification by capillary immunoassay. (A) Densitometry analysis and chemiluminescence signals of the control, (B) densitometry analysis and chemiluminescence signals of samples treated for 1 h with 10 mM ammonium chloride, (C) * indicates significant increase (p<0.05) of ICAM-1 expression. Data are expressed in means±SEM. All experiments were conducted in triplicate.

Determination of eNOS concentration in human brain endothelial cells

The concentration of eNOS in samples was determined in reference to the standard graph using the optical density (OD) obtained from the cell lysate at a wavelength of 450 nm. The eNOS concentration in the control and treated groups are shown in Figure 3. Based on the results, the eNOS concentration in the treatment group (602.5±119.5 pg/mL) increased significantly (p<0.001) compared to the control group (544±66.5 pg/mL).

Figure 3:

Concentration of eNOS in human brain endothelial cells lysate of control and treatment group (triplicate). Treatment group was treated for 1 h with 10 mM ammonium chloride. * indicates significant increase (p<0.001) of eNOS concentration. Data were expressed in means±SEM. All experiments were conducted in triplicate.

Discussion

HBECs are part of the BBB that are crucial in the maintenance of brain permeability. Neurogenerative disorders such as Alzheimer’s disease can cause dysfunctions within the BBB, as degenerated endothelium was observed in the brain vasculature of Alzheimer’s disease patients. This is closely related with the HBEC, as its endothelial dysfunction is increasingly implicated in the development of cardiovascular pathology, neurodegenerative diseases and aging. Recently, lysosomal dysfunction has been highlighted as important in the cellular response to stressor and has been associated to many diseases. The loss of lysosomal autophagy may be one of the mechanisms that elicits functional loss of the HBECs that is contributed by the increase of reactive oxygen species (ROS), inflammation, and loss of tight junction proteins. Thus, this study investigated the cellular changes in HBECs exposed to a lysosome inhibitor through the quantification of claudin-5, ICAM-1 and eNOs that are important molecules in maintain endothelium functions.

In this study, the lysosomotropic agent ammonium chloride, an established lysosomal function inhibitor was used to inhibit the lysosome function in HBECs in order to see their direct effects on these cells through changes in selected protein expressions. Ammonium chloride could penetrate the lysosome, which is an acidic membrane bound organelle and accumulate as protonated forms. This will cause an increase in the intravesicular pH and inhibition of the endosome lysosome system acidification that prevent the normal functioning of the lysosome [17]. In addition, it can prevent the autophagic flux and increased the level of misfolded proteins, that cause elevated stress in the cell [17]. Expression of Claudin-5, ICAM-1 and eNOS were identified in this study and it was found that all parameters in the treated group had significant increases compared to control in a short duration of time.

Claudins are part of membrane proteins built in the TJs of endothelial cells for paracellular pores and barriers. Thus, claudins determine the permeability properties of endothelial cells and has been used as potential markers of BBB integrity [18]. Increased Claudin-5 expression has shown to demonstrate a neuroprotective role in neurodegenerative diseases [19]. HBECs treated with ammonium chloride was initially expected to have reduced Claudin-5 expression due to increased TJ permeability from chemical exposure, as the increase in BBB permeability was due to nitrosative stress, which is a type of oxidative stress [20]. However, previous study on Claudin-5 knockdowns model demonstrated that size-selective opening of the BBB for molecules smaller than 800 Da occurred rather than general breakdown of TJs [21].

Accumulation of aggregated proteins due to exposure to environmental stimuli leads to endothelial stress, this will trigger specific response pathways within cells to encounter the events via clearance of the defective proteins [22]. The endothelial cells will remodel its structure and trigger adaptive changes in endothelial cell signaling, creating a control mechanism to maintain the cellular homeostasis [23]. Another study found that the endocytosis of claudin is essential for the regulation of claudin content in the paracellular barrier [24]. Claudins can be targeted for degradation or for recycling to the plasma membrane. Claudins are endocytosed into endothelial cells in large double membrane vesicles and crossover endocytosed Claudin-5 was found to be increased after inhibition of lysosomal degradation using a lysosome inhibitor by chloroquine [25]. Turnover of Claudin-5 is continuous with the protein having a moderately short in vitro half-life of 90 min [25]. This might suggest that Claudin-5 expression increased depending on the exposure concentration to certain chemicals at certain durations that can determine the extent of damage incurred to endothelial cells. In another study, ammonium chloride has been found to increased claudin level indicating that it is normally rapidly trafficked to lysosomes [26]. Increase of claudin levels could be partly caused by the inhibition of autophagic degradation that is mediated by lysosome [27]. Autophagy activation forms an isolation membrane involved cytoplasmic constituents in a double membrane structure known as autophagosome that will fuse with lysosomes to form an autophagolysosome for the degradation of the molecules or cargo. Lysosome function inhibition could mediate the accumulation of claudin from being degraded.

Lysosome dysfunctions can cause an accumulation of metabolites or autophagosomes that are not degraded within the cells, and this may lead to increased expression marker for inflammation process. The expression of vascular adhesion molecules mediated for the leukocyte transmigration process is minimal or undetectable under physiological conditions and stimulation of inflammation may increase the ICAM-1 expression [28]. In the study, the upregulated ICAM-1 expression was observed after 1 h of treatment compared to the control group. This indicates that brain inflammation has initiated may be due to the accumulation of autophagosomes and non-degradable metabolites in endothelial cells due to lysosome or autophagy dysfunctions. In the early phase of brain inflammation, the neutrophil will interact with endothelium cells by binding to the ICAM-1 and during this process the expression of endothelial cell ICAM-1 increases further [29]. In addition, ICAM-1 can also result in the accumulation of leukocytes and adhesions on endothelium cells that can damage the BBB, allowing inflammatory cells such as granulocytes to infiltrate the brain and release neurotoxic substances that injure neurons [28]. Our finding suggests that inhibition of the lysosome function can induce an early inflammatory response in HBECs through increased ICAM-1 expression.

Endothelial cells play an important role in maintaining the vascular tone by producing endothelial nitric oxide synthase (eNOS) which provides nitric oxide (NO). On the other hand, the bioavailability of NO is impaired in endothelial dysfunction. There are several common regulatory mechanisms involving eNOS activity such as calcium/calmodulin and caveolin, which control activating phosphorylation by AMP-activated protein kinase (AMPK) as well as localization of eNOS [30]. ROS is generated by the accumulation of dysfunctional organelles due to lysosome dysfunctions [31]. A study showed that ROS, such as hydrogen peroxide (H₂O₂), increases the expression of eNOS by regulating the calcium/calmodulin-dependent protein kinase II (CaMKII) mechanism [32]. CaMKII is a serine/threonine kinase that involves in transduction of intracellular Ca²⁺ signals. Bradykinin treatment, an agent that induces Ca²⁺ influx, results in rapid Ca²⁺ influx as well as transient activation of CaMKII and eNOS in human umbilical vein endothelial cells [33]. Therefore, the results from this study might suggest that increased expression of eNOS might serve as a regulatory mechanism at the early exposure of the chemical. These findings might suggest that impaired lysosome dysfunction form the inhibitor could be associated with accumulated of claudin protein yet to be degraded that can give in activation of inflammation response and the ROS produced could stimulate the increase of eNOS formation.

Conclusions

In conclusion, our study showed that HBECs treated with ammonium chloride exhibit increased expression of Claudin-5, ICAM-1 and eNOS, which we suggest may act as early cellular responses to a lysosome inhibitor.