Effect of Cu²⁺ on angiogenesis and nucleoli morphology of cultured endothelial cells of the rat cerebral cortex

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Introduction

Disruption of copper homeostasis is a key pathological feature of several neurodegenerative diseases, including Wilson-Konovalov disease (WKD; also known as Wilson’s disease;), Parkinson’s disease, and Alzheimer’s disease [1–3]. The prevalence of WKD in European countries ranges from 1.2 to 1.8 cases per 100,000 population; however, mutations in the ATP7B gene causing WKD are carried by approximately 1 in 100 individuals [4]. This gene encodes a P-type ATPase, which functions as a transmembrane copper transporter. Mutations in the ATP7B gene disrupt intracellular copper transport in hepatocytes. This results in impaired copper binding to apoceruloplasmin, leading to increased serum concentrations of free copper, its accumulation in various organs and tissues, primarily the liver, brain, kidneys, and cornea. Elevated copper levels in the brain induce morphological changes in glial cells: alterations in astrocyte morphology, formation of intermediate microglial phenotypes, increased microglial size, and oligodendroglial edema. In severe cases, neuronal loss and destruction of myelinated nerve fibers occur, resulting in neuropsychiatric impairments. Conversely, many diseases of the nervous system including WKD can result in damage to the blood-brain barrier [1]. Permeability of the microcirculatory vessels in the brain is impaired in WKD [6]. Furthermore, increased serum levels of intercellular adhesion molecule 1 in WKD patients may indicate disruption of the blood-brain barrier [7]. Pathological changes in the microcirculatory bed are most likely associated with the effect of Cu²⁺ ions on brain endothelial cells (ECs) [8]. At the same time, it is known that copper promotes angiogenesis by playing a crucial role in regulating hypoxia-inducible factor-1, which controls the expression of angiogenic genes [9, 10]. In pathological conditions associated with microcirculatory disorders, such as trauma, stroke, or tumor, angiogenesis in the adult brain can be activated [11]. However, significant accumulation of Cu²⁺ can lead to EC damage [12]. The effect of Cu²⁺ accumulation in the brain on angiogenesis processes remains largely unexplored. In turn, in biomedical research on WKD, there is an urgent need to study changes in microcirculatory vessels, as well as organelle impairments occurring in the ECs themselves. Effective angiogenesis is associated with multiple metabolic processes, including biosynthesis in endothelial cells, as well as with the activity of the cell nucleolus. Interestingly, the protein angiogenin, which can stimulate angiogenesis, is specifically taken up by ECs and transported to the nucleus, where it accumulates in the nucleolus [13]. Therefore, the question of how increased Cu²⁺ concentration affects the nucleolus of ECs is of significant interest. This structure participates in ribosome formation and quality control, which relates to the regulation of protein synthesis [14]. The nucleolus also influences genome organization, the cell cycle, and cellular stress responses [15, 16]. Currently, accumulated data demonstrate that nucleolar stress plays a key role not only in hereditary ribosomopathies but also in other diseases such as Huntington’s disease and parkinsonism [17, 18].

Based on the above, it can be hypothesized that one component of WKD pathogenesis is EC damage due to chronic endogenous intoxication resulting from impaired intracellular copper transport [19, 20]. We proposed that excess copper ions may disrupt angiogenesis and induce changes in the nucleolus of ECs. Testing this hypothesis constituted the primary aim of this study.

Materials and Methods

The study utilized monolayer dissociated EC cultures derived from the cerebral cortex of Wistar rats.

Protocols were approved by the Ethics Committee of Russian Center of Neurology and Neurosciences (Protocol No. 9-4/23 dated 23 November, 2023). All isolation and plating techniques complied with ethical standards established by Russian Federation legal acts, principles of the Basel Declaration, and ethical guidelines for animal research.

Endothelial cells cultures were obtained using a previously described method [21, 22]. The cerebral cortex was extracted from anesthetized 14-day-old rats and rolled over sterile filter paper to remove meninges. After removing large blood vessels and white matter, the cerebral hemispheres were washed multiple times with cold Hank’s solution (Gibco Life Technologies), minced, and centrifuged for 5 min at 1000 rpm. Bovine serum albumin (25%, Sigma) was added to the pellet in a volume twice that of the pellet, followed by 25–30 pipetting cycles using up-and-down motions with a Pasteur pipette. The homogenate was centrifuged for 10 min at 2000 rpm. The supernatant was pipetted again 25–30 times and centrifuged. A 0.1% collagenase solution (Sigma-Aldrich) in phosphate buffer (Gibco Life Technologies) was added to the pellet and incubated for 40 min at 37°C. The digested microvessels were centrifuged for 5 min at 1000 rpm. The pellet was resuspended in culture medium containing 80% modified Eagle’s medium (DMEM-F12, Gibco Invitrogen Corporation), 20% fetal bovine serum (HyClone), 2 mM glutamine (GlutaMAX, Gibco), 16.6 mM glucose, 1.4 μM hydrocortisone, and 1 mM pyruvate, then plated on a 40-mm plastic Petri dish (Medpolymer) coated with Matrigel at a density of 1–2 × 10⁵ cells/mL. Cultures were maintained until a confluent monolayer formed over one week, with two medium changes. Cells were then subcultured into polylysine-coated 96-well plates at the same density. Experiments used first-passage cells. The method employs extensive pipetting and centrifugation, which eliminates most glial cells, eliminating the need for chemical suppression of astrocyte growth [20].

Cell viability after experiments was assessed using a colorimetric MTT test based on the reduction of a yellow water-soluble tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) to purple formazan: brighter cell staining indicates higher culture survival rates. For this purpose, cultures were incubated with 0.5 mg/ml MTT for 30 minutes. Next, the culture medium was aspirated, and 50 µl of dimethyl sulfoxide was added to each well. Photometry was performed with a microplate reader (SpectraMax M2, Molecular Devices) at 570 nm. Control culture viability was set at 100%, and treated cell viability was calculated as a percentage of the control [23, 24].

The Angiogenesis Assay kit (Abcam, ab204726) was used according to the manufacturer’s instructions. Fifty microliters of matrix were dispensed into wells of a 96-well plate and incubated for 1 hour in a thermostat. Uncoated wells were used for control. Endothelial cells were seeded onto the coating according to the above protocol at a density of 2 × 10⁵ cells/mL. Live observations of culture conditions were conducted using phase-contrast microscopy with an inverted microscope (Olympus CKX41). Imaging and photograph acquisition were performed using the EVOS M7000 imaging system (ThermoFisher Scientific) in automated mode, capturing 95% of the well area. The length of formed tubes/processes and the number of closed loops resulting from endothelial angiogenesis were assessed.

To stain nucleoli, cultured ECs were fixed with 5% formaldehyde in PBS (Sigma) for 15 minutes, washed with PBS, and incubated in acridine orange solution (0.74 mg/mL, Sigma-Aldrich) for 10 minutes. Nucleoli were visualized using an Olympus IX71 confocal microscope with a spinning disk, a ×100 objective, and a 488-nm OBIS laser controlled by Coherent Connection 3 software. Fluorescent images of cells for nucleoli visualization were acquired at an emission wavelength of 628 nm. Morphometric analysis of nucleoli was performed using Fiji software.

The results were processed using Statistica v. 13.3 software (StatSoft Inc.) by one-way ANOVA with Newman-Keuls post-hoc test or unpaired t-test. Data are presented as mean ± standard error of the mean (SEM). The data were considered statistically significant at р < 0.05. Each experiment was repeated at least 3 times.

Results

A study of cultured rat cerebral cortex ECs using phase-contrast microscopy demonstrated that pronounced cell damage in the monolayer is observed when the Cu²⁺ concentration in the culture medium reaches 200–300 μM (Fig. 1, A–C). However, when determining toxic concentrations of Cu²⁺ in vitro, a decrease in formazan production was shown in cells incubated with MTT at Cu²⁺ concentrations of 100 μM and higher in the culture medium (Fig. 1, D), indicating reduced cell survival in the culture.

 

Fig. 1. The toxic effect of Cu²⁺ on cultured rat cerebral cortex ECs.

А–С: phase contrast. A: control; B: 200 μM Cu²⁺; C: 300 μM Cu²⁺; D: formazan formation via MTT metabolism (viability). *p < 0.05 vs. control (0 μM Cu²⁺).

 

However, we previously demonstrated impairment of barrier function in cultured ECs at 50 μM Cu²⁺ in the culture medium [12]. Here, we show that Cu²⁺ substantially disrupts angiogenesis in rat cerebral cortex-derived EC cultures. Using the Angiogenesis Assay Kit (which models key stages (EC migration and differentiation) in vitro), we found that 100 μM Cu²⁺ significantly reduced closed-loop formation and total process length to 52 ± 7% and 78 ± 4%, respectively (Fig. 2).

 

Fig. 2. Cu²⁺ disrupts angiogenesis in rat cerebral cortex-derived EC cultures.

Phase-contrast microscopy: (A) control culture; (B) after 24 h at 100 μM Cu²⁺. Quantitative data: (C) closed loops on matrix; (D) total process length.

 

Fig. 3. Morphological changes of the nucleolus in cultured ECs under toxic effects of CuCl₂.

Microphotograph of nuclei in cultured ECs under control conditions (A) and in the presence of 200 μM Cu²⁺ (B). Staining with acridine orange. Nucleoli are indicated by arrows. Scale bar: 10 μm. Quantitative assessment of nucleolar area (C) and EC nuclear area (D).

 

Higher Cu²⁺ concentrations induced nucleolar alterations in EC nuclei.

Morphometry of acridine orange-stained EC nucleoli revealed that 200 μM Cu²⁺ exposure for 24 h increased nucleolar profile area from 1.33 ± 0.07 to 1.71 ± 0.09 μm², while nuclear size remained unchanged vs. controls (Fig. 2).

Discussion

Our results support the hypothesis of toxic effects of Cu²⁺ (high concentrations) on ECs, mediated by nucleolar apparatus damage and leading to suppression of angiogenic activity. However, it should be noted that the study by T. Litwin et al. demonstrated that Cu²⁺ concentration in the brains of individuals with WKD is approximately 41.0 ± 18.6 μg/g dry weight, whereas in healthy individuals it is 5.4 ± 1.8 [19, 24]. Nevertheless, correlating in vivo and in vitro data is quite challenging, and moreover, WKD involves a pathogenic mutation in the ATP7B, gene encoding the Cu²⁺ transporter. Alterations in the structure of the protein encoded by this gene and/or reduced enzymatic activity are associated with impaired intracellular Cu²⁺ transport [25]. Therefore, we relied on Cu²⁺ concentrations used in studies performed on EC cultures [8, 12]. Interestingly, suppression of the Cu²⁺ exporter ATP7B activity in brain microvascular ECs results in loss of barrier function in hBMEC [26]. Phase-contrast microscopy revealed that significant cell damage in the monolayer was observed when the Cu²⁺ concentration in the culture medium reached 200–300 μM. However, the MTT assay showed that formazan accumulation in cells incubated with MTT significantly decreased at Cu²⁺ concentrations in the culture medium of 100 μM and higher. Currently, tetrazolium salts have become one of the most widely used tools in cell biology for measuring cellular metabolic activity. Mammalian cell fractionation studies indicate that NADH is responsible for the majority of MTT metabolism, with the bulk of cellular MTT metabolism occurring outside the mitochondrial inner membrane and involving NADH- and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors [27]. Thus, MTT metabolism is associated not only with mitochondria but also with the cytoplasm and non-mitochondrial membranes, including the endosomal/lysosomal compartment and plasma membrane [28].

Cu²⁺ at low concentrations promotes angiogenesis; however, this effect is inverted under supraphysiological concentrations of Cu²⁺: for instance, at concentrations around 100 μM, the angiogenic EC activity in vitro is suppressed. We are the first to demonstrate that the anti-angiogenic activity of Cu²⁺ ions is associated with disruptions in the nucleolar apparatus of ECs. Nucleoli are dynamic structures formed around clusters of ribosomal DNA. Components of this organelle include ribosomal DNA, pre-ribosomal RNA, ribosomal RNA, transcription factors, RNA- and DNA-binding proteins, small non-coding RNAs, and numerous proteins with currently unknown functions [29]. It is known that ultraviolet and γ-radiation, oxidative stress, exposure to genotoxic chemotherapeutic agents, hypoxia, as well as nutrient and growth factor deficiencies can induce nucleolar stress — a set of structural and functional changes in the nucleolus arising from disruptions in rRNA transcription by RNA polymerase I, processing, and/or assembly of ribosomal subunits. Collectively, the decrease in mitochondrial membrane potential in ECs [12] and the morphological changes in nucleoli under Cu²⁺ exposure, as shown in the present study, suggest that Cu²⁺ -induced damage to ECs is accompanied by nucleolar stress.

Conclusion

Excess Cu²⁺ ions suppress angiogenic activity and may induce nucleolar stress in cerebral endothelial cells in vitro. This may represent a universal cellular response associated with ribosomal quality control and regulation of biosynthesis in the cell. Likely, the nucleolar apparatus of ECs could be considered a potential target for pharmacotherapy aimed at restoring the angiogenic potential of cerebral microvascular ECs.

About the authors

Elena V. Stelmashook

Russian Center of Neurology and Neurosciences

Author for correspondence.
Email: annaly-nevrologii@neurology.ru
ORCID iD: 0000-0003-2533-7673

Dr. Sci. (Biol.), leading researcher, Laboratory of neurobiology and tissue engineering, Brain Institute

Russian Federation, Moscow

Elizaveta E. Genrikhs

Russian Center of Neurology and Neurosciences

Email: annaly-nevrologii@neurology.ru
ORCID iD: 0000-0002-3203-0250

Cand. Sci. (Biol.), senior researcher, Laboratory of neurobiology and tissue engineering, Brain Institute

Russian Federation, Moscow

Olga P. Alexandrova

Russian Center of Neurology and Neurosciences

Email: annaly-nevrologii@neurology.ru
ORCID iD: 0009-0006-9109-1463

Cand. Sci. (Biol.), researcher, Laboratory of neurobiology and tissue engineering, Brain Institute

Russian Federation, Moscow

Anna B. Strizhkova

Russian Center of Neurology and Neurosciences; M.V. Lomonosov Moscow State University

Email: annaly-nevrologii@neurology.ru
ORCID iD: 0009-0006-5434-963X

student, Department of cell biology and histology, Faculty of biology, laboratory researcher, Laboratory of neurobiology and tissue engineering, Brain Institute

Russian Federation, Moscow; Moscow

Alina E. Lapieva

Russian Center of Neurology and Neurosciences; M.V. Lomonosov Moscow State University

Email: annaly-nevrologii@neurology.ru
ORCID iD: 0009-0008-9091-6974

student, Department of cell biology and histology, Faculty of biology, laboratory researcher, Laboratory of neurobiology and tissue engineering, Brain Research Institute

Russian Federation, Moscow; Moscow

Marina R. Kapkaeva

Russian Center of Neurology and Neurosciences

Email: annaly-nevrologii@neurology.ru
ORCID iD: 0000-0002-2833-2897

researcher, Laboratory of neurobiology and tissue engineering, Brain Institute

Russian Federation, Moscow

Nickolay K. Isaev

Russian Center of Neurology and Neurosciences; M.V. Lomonosov Moscow State University

Email: annaly-nevrologii@neurology.ru
ORCID iD: 0000-0001-8427-1163

D. Sci. (Biol.), leading researcher, Laboratory of neurobiology and tissue engineering, Brain Institute, Associate Professor, Department of cell biology and histology, Faculty of biology

Russian Federation, Moscow; Moscow

References

Supplementary files