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. Author manuscript; available in PMC: 2022 Jan 20.
Published in final edited form as: J Endod. 2020 Jun 24;46(8):1113–1119. doi: 10.1016/j.joen.2020.04.005

Moderately Acidic pH Promotes Angiogenesis: An In Vitro and In Vivo Study

Mohammad Ali Saghiri *,†, Armen Asatourian ‡, Steven M Morgano *, Shoujian Wang §, Nader Sheibani §,‖
PMCID: PMC8774255  NIHMSID: NIHMS1768853  PMID: 32593435

Abstract

Introduction:

This study evaluated the effect of different pH values of 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4 on angiogenesis.

Methods:

Endothelial cells were isolated from the mice molar teeth and placed in 42 Matrigel (Corning, NY)-coated wells, which were prepared and divided into 6 groups (n = 7). Synthetic tissue fluid was prepared and divided into 6 parts, and their pH values were adjusted to 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4. A 2-mL volume from each group was diluted in the growth medium at a ratio of 1:3 and used for tubulogenesis assay. Forty-two 6-week-old mice in 6 groups (n = 7) were used for choroidal neovascularization (CNV). A 2-μL volume from each group or saline (control) was delivered by intravitreal injection on the day of laser application and 1 week later. Data on the number of nodes, the total length of the branches, and CNV areas (μm2) were determined using ImageJ software (National Institutes of Health, Bethesda, MD) and analyzed with 1-way analysis of variance and post hoc Tukey tests. The correlation was assessed between the tested variables.

Results:

The number of nodes decreased with changes in pH values as follows: 6.4 > 5.4 > 7.4 > 8.4 > 9.4 > 4.4. The total branch length decreased with pH value changes as follows: 6.4 > 4.4 > 6.4 > 7.4 > 8.4 > 9.4, and the CNV areas decreased with pH value changes as follows: 6.4 > 5.4 > 4.4 > 7.4 > 8.4 > 9.4.

Conclusions:

Moderately acidic pH values (5.4 and 6.4) enhanced angiogenesis, whereas moderately alkaline pH values (8.4 and 9.4) suppressed angiogenesis.

Keywords: Angiogenesis, choroidal neovascularization, endothelial cells, Matrigel, tubulogenesis

Angiogenesis is a vital process in the regeneration and survival of all of the biological tissues. It is responsible for the formation of new blood vessels from preexisting capillaries under both physiological and pathologic conditions1,2. The alteration or modification of angiogenesis is the basic target for several treatment modalities in the medical field. These treatment modalities consist of stimulation of angiogenesis in peripheral arterial disease, cardiovascular diseases, or disorders in wound healing3,4 or inhibition of angiogenesis for limiting the growth of cancerous tumors5–7. Angiogenesis is also of the utmost importance in the health of the dental pulp, and its enhancement promotes the regeneration of the dentin-pulp complex8.

After signaling for the initiation of angiogenesis because of the insufficiency of oxygen, several hemodynamic factors including growth factors, cytokines, transcription factors, endogenous inhibitors of angiogenesis, adhesion molecules, matrix metalloproteinases (MMPs), and components of the extracellular matrix (ECM) are required for angiogenesis to occur9–13. In addition, the pH value of the subjected biological tissue might affect angiogenesis14. However, there are little and inconsistent data regarding the effect of pH on angiogenesis. Previous studies showed that low pH values could not stimulate the macrophages to secrete proangiogenic factors at wound sites15 and down-regulated angiogenesis by affecting endothelial cells (ECs)16. However, extracellular acidic pH could stimulate an angiogenic response14 and promote vasculogenesis events17.

The pH value might also have a crucial role in the function of the dental pulp cells because it was indicated that the pH could affect the mineralization ability of human dental pulp cells18, and an acidic pH might induce proangiogenic activities in isolated pulp ECs19. However, it is still not clear whether an acidic pH has promoting or inhibitory effects on angiogenesis. Furthermore, the effect of an alkaline pH on the angiogenesis is not yet well discussed. Hence, the present study evaluated the effect of different pH values (4.4, 5.4, 6.4, 7.4, 8.4, and 9.4) on angiogenesis. It was hypothesized that changes in the pH value of the environment can modify angiogenesis and regeneration processes.

MATERIALS AND METHODS

Forty-two samples were selected and divided into 6 groups (n = 7) for each of the study models.

Preparation of Various pH Solutions

This step was performed according to a previously described method20. First, we prepared synthetic tissue fluid (STF) by using 1.7 g KH2PO4, 11.8 g Na2HPO4, 80.0 g NaCl, and 2.0 g KCl (pH = 7.4). This STF solution was divided into 6 parts, and for the acidic pH groups (4.4, 5.4, and 6.4), the pH of the STF was adjusted using butyric acid (pH = 4.4), and for the alkaline pH groups (8.4 and 9.4), the STF pH was adjusted using potassium hydroxide (pH = 10.4). For the control group, normal saline (pH = 7.4) was used. A 2-mL volume of each prepared pH solution was diluted with the low glucose EC medium at a ratio of 1:3 (1 part STF solution to 3 parts medium) in a 15-mL tissue culture tube and incubated for 1 day at 37°C and 98% humidity. In the control group, normal saline solution was mixed with the growth medium. The prepared solutions were used for EC tubulogenesis assay on Matrigel (Corning, NY), and the laser-induced choroidal neovascularization (CNV) studies were performed as detailed later.

Matrigel Tubulogenesis Assays

The molar dental pulp ECs were isolated according to methods described previously21,22 and used for the tubulogenesis assay. The tubulogenesis assays were performed according to a method described previously by Saghiri et al22. Briefly, all the 42 Matrigel-coated wells were prepared and seeded with molar ECs at 2500 cells/cm2 in 2 mL of the solutions with adjusted pH values and incubated for 72 hours. The density of the cells for each experimental group was compared with the control medium. The medium was replaced every 24 hours, and after 72 hours, the arrays were washed with serum-free medium and stained with a 5-mmol/L Cell Tracker Green (Invitrogen, Carlsbad, CA) solution prepared in the serum-free medium for 45 minutes. After 15 minutes of staining, the arrays were washed with the serum-free medium and incubated for 30 minutes in the growth medium. The arrays were then washed with phosphate-buffered saline (PBS) and fixed for 30 minutes in 10% buffered formalin (Fisher, Hanover Park, IL). The wells were incubated in the PBS overnight and photographed using a Nikon TE300 fluorescence microscope (Nikon, Tokyo, Japan) within 48 hours of fixation.

CNV Studies

CNV was performed according to the methods described by Wang et al23. CNV was induced in 6-week-old, C57BL/6J female mice (Jackson Laboratories, Bar Harbor, ME) by laser photocoagulation-induced rupture of the Bruch membrane on day 0. The mice were anesthetized using ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg), and the pupils were dilated using tropicamide ophthalmic solution. Laser photocoagulation (75-μm spot size, 0.1-second duration, and 120 mW) was performed in the 9, 12, and 3 o’clock positions of the posterior pole of each eye with the slit lamp delivery system of an Oculight GL diode laser (Iridex, Mountain View, CA) and a handheld coverslip as a contact lens to view the retina. The prepared solutions with different pH values and the control groups were delivered by intravitreal injection (2 μL/eye) on the day of laser application and 1 week later (Fig. 1). After 14 days, the eyes were removed and fixed in 4% paraformaldehyde at 4°C for 2 hours. After 3 rounds of wash in PBS, the eyes were sectioned at the equator, and the anterior half, vitreous, and retinas were removed. The remaining eye tissues were incubated in a blocking buffer solution (5% fetal calf serum and 20% normal goat serum in PBS) for 1 hour at room temperature followed by incubation with anti–ICAM-2 (1:500 in PBS containing 20% fetal calf serum and 20% normal goat serum; BD Biosciences, San Jose, CA) overnight at 4°C. The remaining eye tissues were then washed 3 times with PBS and incubated with the appropriate secondary antibody. The retinal pigment epithelium-choroid-sclera complex was subjected to 5 to 6 releasing radial incisions and flat mounted on a slide with Vecta Mount AQ (Vector Laboratories, Inc, Burlingame, CA). The samples were viewed under a fluorescent microscope, and the images were captured in the digital format using a Zeiss (Oberkochen, Germany) microscope (Fig. 2D).

FIGURE 1 –

FIGURE 1 –

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A schematic representation of the laser-induced CNV model in mice.

FIGURE 2 –

FIGURE 2 –

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The box plots of the means and standard deviations of (A) the number of nodes, (B) total branch length, and (C) CNV surface area (μm2) and of the control and experimental groups. (D) Schematic skeletonization of ImageJ software for a representative sample.

Image and Statistical Analysis

The number of nodes, total branch length, and surface area (in μm2) of CNV were measured using ImageJ software (National Institutes of Health, Bethesda, MD) based on the methods previously described by Saghiri et al19. Briefly, the images were opened, inverted, set scaled, and converted into an 8-bit file. Eventually, the images were smoothed by the “unsharp mask” feature, and the number of nodes, total length branch, and CNV surface area were determined. The data were analyzed by Kolmogorov-Smirnov, 1-way analysis of variance (ANOVA), and post hoc Tukey tests at a significance level of P < .05. In addition, the correlation measurements were performed between the test-dependent variables.

RESULTS

Matrigel Tubulogenesis Assays (Number of Nodes and the Total Branch Length)

One-way ANOVA showed significant differences between the data from the groups for the number of nodes and total branch length (P < .001). The means ± standard deviations of the number of nodes for the control and experimental pH groups were as follows: 75.86 ± 5.46 (pH = 4.4), 162.57 ± 35.0 (pH = 5.4), 169.71 ± 30.8 (pH = 6.4), 147.71 ± 33.8 (pH = 7.4), 109.4 ± 15.32 (pH = 8.4), and 93.1 ± 10.38 (pH = 9.4). According to post hoc Tukey tests, there were significant differences in the mean number of nodes between the pH values of 4.4, 8.4, and 9.4 and 5.4, 6.4, and 7.4 (P < .001), whereas no significant differences were noticed between the 4.4, 8.4, and 9.4 groups or between the 5.4, 6.4, and 7.4 groups (P > .05) Fig. 2A).

The means ± standard deviations of the total branch lengths of the control and experimental groups were as follows: 1275.86 ± 104.6 (pH = 4.4), 1288.29 ± 90.201 (pH = 5.4), 1310.86 ± 85.81 (pH = 6.4), 1263.14 ± 80.57 (pH = 7.4), 990.43 ± 45.67 (pH = 8.4), and 793.4 ± 63.46 (pH = 9.4). One-way ANOVA showed significant differences between the control and treatment groups in the total branch length (P < .001). According to post hoc Tukey tests, significant differences were found between pH values of 4.4, 5.4, 6.4, 7.4, and 8.4 (P < .001); however, no significant differences were found between 8.4 and 9.4 pH values (P > .05) (Fig. 2B).

CNV Analysis

The means ± standard deviations of the CNV area of the control and experimental groups were as follows: 914.29 ± 96.211 (pH = 4.4), 1244.57 ± 227.75 (pH = 5.4), 1150.57 ± 228.8 (pH = 6.4), 727.71 ± 67.5 (pH = 7.4), 641.0 ± 64.93 (pH = 8.4), and 444.7 ± 55.7 (pH = 9.4). One-way ANOVA showed significant differences between the control and treatment groups (P < 0.001). Significant differences were noted between the pH values of 5.4 and 6.4 and the other groups (P < 0.001; however, no significant difference was found between the pH values of 8.4 and 9.4 (P = .139) (Figs. 2C and 3A–F).

FIGURE 3 –

FIGURE 3 –

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The images of CNV analysis of the experimental groups: (A) pH = 7.4, (B) pH = 4.4, (C) pH = 5.4 (please note the arrow showing the formation of blood vessels), (D) pH = 6.4 (please note the arrow showing extensive networklike blood vessel formation), (E) pH = 8.4, and (F) pH = 9.4. In the CNV images, only at pH = 6.4 and 5.4 was extensive blood vessel formation noticed, which was more prominent at pH = 6.4.

The analysis of correlation showed a significant correlation between the data of the number of nodes, the total branch length, and the CNV surface areas. This is consistent with the observed P value of < .001.

DISCUSSION

In our in vitro model, we used the Matrigel tubulogenesis assay, which is an accepted and widely used method for the evaluation of angiogenesis in vitro24. In our in vivo studies, we used the laser-induced CNV model, which is also a widely used method for screening and evaluation of angiogenesis25–27. Our previous studies showed that the isolated ECs from the mice dental pulp, especially from the molar teeth, can be regarded as a reliable cell line for the evaluation of the effect of different materials or conditions on angiogenesis19,22. The dilution of pH solutions with EC medium was performed similar to that in our previous study in order to compare our findings with those of our previous study19.

We noticed that a moderately acidic pH value (5.4 and 6.4) promoted angiogenesis as evident by the increased number of nodes, the total branch length, and the CNV area compared with the neutral pH of 7.4 (Figs. 2 and Fig. 3C and D), which is consistent with the results of previous studies. Xu et al28 showed that the acidic pH of ECM increased the levels of soluble vascular endothelial growth factor (VEGF) in glioblastoma cells, enhancing angiogenesis through activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling pathway. In another study, Mena et al17 showed that the acidic preconditioning of endothelial colony-forming cells promoted vasculogenesis. However, in a study by Faes et al16, the acidic pH of ECM reduced EC proliferation and migration, which can negatively affect tubulogenesis.

These differences in outcomes might be justified by the fact that the extracellular acidic pH of the environment surrounding the vascular network has 2 distinct effects on angiogenesis. The first is the direct effect, which was claimed to be a negative influence on the proliferation and migration of EC16. However, the second effect is the indirect effect of the acidic pH, which appears to be a proangiogenic influence and is through the changes made inside the ECM components in vivo or the other EC surrounding environment, such as Matrigel in in vitro conditions. This effect was also explained by Burbridge et al14, who claimed that the low environmental pH is not necessarily detrimental to the angiogenesis of ECs, and the moderate acidic pH (pH = 6.9) could stimulate angiogenesis through the release of high concentrations of exogenous growth factors29. In other words, the acidic pH has a similar role as the MMPs, which release the exogenous growth factors by degrading the ECM. The same events occur during tumor angiogenesis in which the metabolic activities of cancerous cells lead to hypoxia; an acidic environment; and the production of several growth factors such as VEGF, basic fibroblast growth factor, and platelet-derived endothelial cell growth factor. These growth factors can be released by the acidic pH and MMPS inside the ECM and promote angiogenesis in addition to the hypoxia-driven angiogenesis29. Similar growth factors could be found in the EC culture medium, which was added to Matrigel for the evaluation of angiogenesis. The weak or moderate acidic pH (5.4 and 6.4) led to the release of these growth factors in Matrigel and stimulation of angiogenesis through an indirect effect. However, the stronger acidic pH (4.4) diminished angiogenesis because of the direct negative effects on the EC, as mentioned previously (Fig. 2).

The present study also showed that alkaline pH could significantly reduce tubulogenesis and neovascularization. To the best of our knowledge, no study in the literature has evaluated the effect of extracellular alkaline pH on ECs and angiogenesis. The results of the present study can be explained by the denaturing and deteriorating effect of alkaline pH on proteins. It was previously shown that alkaline pH causes structural changes in proteins30. However, there is still a lack of information regarding the effect of extracellular alkaline pH on angiogenesis and EC function. In addition, the mechanism of action of the detrimental effect of alkaline pH on angiogenesis is still unclear, and future in vitro and in vivo studies are required to address this issue further. Regarding the correlation between the tested variable, we noticed that the tubulogenesis and neovascularization processes were strongly connected and correlated with each other.

The outcomes of present studies can be related to endodontic biomaterials including vital pulp therapy materials and irrigating solutions. In a previously performed investigation, we showed that both calcium hydroxide (Ca[OH]2) and mineral trioxide aggregate (MTA) have a minimal antiangiogenic effect in direct contact with ECs19. However, several studies reported that Ca(OH)2 and MTA have proangiogenic effects31,32. These differences can be explained by the indirect effects of Ca(OH)2 and MTA. It was indicated that Ca(OH)2 can promote the release of growth factors such as the fibroblast growth factor, transforming growth factor beta, insulinlike growth factor, and platelet-derived growth factor from the dentin matrix31. A similar indirect proangiogenic effect was shown regarding MTA because it was reported that MTA cement can increase the release of proangiogenic factors such as VEGF, transforming growth factor beta 1, interleukin 1 beta, and interleukin 832.

Among endodontic irrigating solutions, sodium hypochlorite and chlorhexidine have an alkaline pH33,34, and EDTA has an average pH of 7.335. In a review study, it was reported that sodium hypochlorite and CHX have antiangiogenic effects, whereas EDTA has a proangiogenic effect because of its effect on the dentin matrix and the release of growth factors32. The outcomes of present studies are consistent with these findings and might also explain the proangiogenic effect of EDTA because a pH of 7.4 promotes angiogenesis. However, in clinical and in vivo conditions, there are several other environmental factors such as the ECM or other cells that can alter the path of angiogenesis.

CONCLUSIONS

Tubulogenesis and neovascularization can be regulated under different pH conditions. A moderate or weak extracellular acidic pH (5.4 and 6.4) stimulates tubulogenesis and neovascularization. This proangiogenic effect is more an indirect effect through the release of extracellular growth factors surrounding the endothelial cells. A stronger extracellular acidic pH (4.4) and alkaline pH (8.4 and 9.4) diminish tubulogenesis and neovascularization.

SIGNIFICANCE.

Endodontic biomaterials that have moderate acidic pH can be regarded as proangiogenic materials. These materials can induce angiogenesis and enhance the vascularization of the dental pulp, which is a determinant factor in the survival of dental pulp after dental carries and traumas.

ACKNOWLEDGMENTS

This publication is dedicated to the memory of Dr. H. Afsar Lajevardi36, a legendry pediatrician (1953–2015). We will never forget Dr. H Afsar Lajevardi’s kindness and support.

This publication is supported by a unrestricted award from the Research to Prevent Blindness to the Department of Ophthalmology and Visual Sciences, Retina Research Foundation (NIH grant nos. P30 EY016665, P30 CA014520, EPA 83573701, EY022883, and EY026078). N.S. is a recipient of RPB Stein Innovation Award. M.A.S. is a recipient of New Jersey Health Foundation Innovation Award.

The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the affiliated organizations. The authors hereby announce that they have had active cooperation in this scientific study and preparation of the present manuscript. The authors confirm that they have no financial involvement with any commercial company or organization with direct financial interest regarding the materials used in this study.

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