Zinc-containing Mohs’ paste affects blood flow and angiogenesis suppression

Abstract

Purpose

Mohs’ paste, which is composed of zinc chloride and zinc oxide starch, is used for hemostasis of superficial malignancy in the clinical setting. We investigated the concentration of intramuscular zinc in mice after Mohs’ paste application and evaluated its relationship with angiogenesis from the perspective of blood flow levels within 24 h.

Methods

Male C57BL/6JJmsSlc mice were administered single dose of Mohs’ paste at 25%, 50%, and 75% after unilateral hind limb surgery, and glycerin, a viscosity modifier, was administered to the control group (0%). Hind limb blood flow levels were measured with a laser Doppler perfusion imaging system (n = 6). The amounts of intramuscular zinc and vascular endothelial growth factor-A (VEGF-A) expression were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) and western blotting, respectively (n = 5 or 3).

Results

Blood flow levels were significantly decreased in the 50% group after 8 h, and significantly decreased in the 25% and 50% groups after 24 h. Intramuscular zinc was significantly increased in the 50% and 75% groups after 8 h. Western blotting showed that VEGF-A levels were significantly increased in the 25% and 50% groups after 8 h. Based on analytical experiments and biological investigation, we predicated the pharmacological effect of Mohs’ paste and found over 50% of it is critical in the blood flow and angiogenesis suppression after more than 8 h of its application.

Conclusions

The results suggest that the mechanism of blood flow suppression is independent of VEGF-A levels and might suppress future angiogenesis. Our findings support that of previous studies, in which Mohs’ paste was expected to induce hemostasis and suppress angiogenesis. It is an excellent ointment that facilitates hemostasis by suppressing blood flow regardless of angiogenesis, and may be apt for situations where hemostasis is required in the clinical setting.

Graphical abstract

Introduction

Angiogenesis plays an important role in homeostasis in humans and animals; its high activity is observed under ischemic conditions or in tumors. Superficial cancerous tissues, such as cutaneous tumors or cutaneous infiltration in breast cancer, are vulnerable, sensitive to damage, and rapidly create new blood vessels. Diffuseness or persistent bleeding after direct and/or indirect contact with a lesion may result in exsanguination from fragile vessels; thus, hemostasis is required [1]. In the 1930s, Frederic F Mohs developed Mohs’ paste containing zinc chloride for the chemical fixation of a cutaneous tumor [2, 3]. However, the original Mohs’ paste included constituents such as stibnite and Sanguinaria canadensis, which are difficult to obtain in Japan. Therefore, its use is limited in Japan. Ohkubo et al. reported a modified Mohs’ paste composed of zinc chloride and zinc oxide starch in 2000, which considerably enhanced the feasibility of its application in the clinical setting [4]. Presently, Mohs’ paste is widely used to treat inoperable superficial cancer or cutaneous infiltration in breast cancer, and controls the exudation, odor, bleeding, and pain from wounds, as well as considerably improves a patient’s quality of life (QOL). Mohs’ paste is involved in hemostasis due to tissue fixation. Zinc chloride ionized by water on the wound surface can aggregate proteins of cancer cells and cancer vessels [5], denature the cell membranes of secondary-infected bacteria on the ulcer surface, and has antibacterial effects against drug-resistant bacteria [6, 7]. In a case report, Mohs’ paste was used for the effective treatment of bleeding and exudation from wounds caused by cutaneous infiltration of breast cancer in the clinical setting [8, 9]. Additionally, Yanazume et al. clearly provided evidence that a single application of Mohs' paste could, without any complications, achieve complete hemostasis in a case of fatal genital bleeding of the uterine cervix; however, the mechanisms of the hemostatic effect have not been reported yet [10].

Zinc is an important trace metal in the body, and its dynamics in humans and animals have been well studied. Generally, it enters the organism through dietary intake and plays a critical role in many biological processes. After being distributed to each organ, zinc is involved in stabilization of the structure of numerous proteins, including cell proliferation and growth, development and differentiation, metabolism, RNA transcription, and DNA synthesis [11, 12]. Furthermore, Chasapis et al. reported that zinc is critical for wound healing, taste acuity, immune system function, prostaglandin production, and blood clotting [13]. Moreover, it has been shown to function as a modulator of synaptic neurotransmission in the forebrain and is reported to be involved in the growth and maturation of neurons [14]. The pharmacological effect of zinc is known to be involved in angiogenesis, which reacts to ischemic conditions in humans and animals. Vascular endothelial growth factor (VEGF) is the main factor enhancing angiogenesis and can be classified as VEGF-A, VEGF-B, VEGF-C, or VEGF-D, and plays an important role in the pathophysiology of diseases such as cancer and ischemia [15, 16]. In particular, VEGF-A is essential for organ remodeling and diseases that involve blood vessels, such as wound healing, tumor angiogenesis, diabetic retinopathy, and age-related macular degeneration [17, 18]. Recent studies have shown that zinc promotes the expression of VEGF and induces the expression of VEGF-mediated signal transduction, such as VEGF receptors 1 and 2 [19, 20]. Moreover, zinc is required for the structural stability of zinc finger proteins (ZFPs), which are transcription factors. It is reported that ZFPs induce the expression of native VEGF isoforms, which leads to the development of a physiologically normal vasculature [21]. However, it has been reported that zinc directly protects endostatin, which is an anti-angiogenic factor, and consequently suppresses angiogenesis [22]. Boehm et al. reported that zinc binding protects endostatin at the N-terminus from proteolytic degradation and its subsequent inactivation [23]. Therefore, zinc-and zinc-related proteins are affected by angiogenetic and anti-angiogenic effects. In contrast, zinc is also known to be involved in hemostasis, and displayed a defective platelet aggregation response to adenosine diphosphate (ADP) and arachidonate in human volunteers [24]. Additionally, zinc-dependent platelet aggregation has been reported [25]. The present study investigated the effects of Mohs’ paste application on angiogenesis, focusing on the recovery of blood flow from the ischemic state in the hind limb, and demonstrated the suppression of angiogenesis through the evaluation of intramuscular zinc concentrations and VEGF-A levels. We selected different concentrations of Mohs’ paste for blood flow analysis and quantification of zinc or biological investigation to compare our findings with that of a previously conducted biological analysis, which had revealed decreased inducible nitric oxide synthase (iNOS) levels upon 50% and 75% Mohs’ paste application [9]. Additionally, we investigated the short time (2–24 h) effect on low-concentration Mohs’ paste (25%). Overall, 50% Mohs’ paste was used in this study.

Materials and methods

Mohs’ paste preparation

Zinc chloride was purchased from Nikko Pharmaceutical Co., Ltd., Gifu, Japan, and zinc oxide starch was purchased from Maruishi Pharmaceutical Co., Ltd., Osaka, Japan. Mohs’ paste was prepared according to a previously described method [9]. Approximately 500 g of zinc chloride was added to 250 mL of distilled water. After dissolution, 250 g of zinc oxide starch was gradually mixed until a saturated solution was obtained using a mortar and pestle, and 20 mL of glycerin was added to adjust the viscosity. This paste was used as the 100% standard paste, and 375, 250, and 125 g of zinc chloride were added to 250 g of zinc oxide starch as 75%, 50%, and 25% Mohs’ paste, respectively. Glycerin was used as the control (0%). The prepared paste was sealed in a plastic container and stored at 4 °C until further use.

Animals

Forty-eight male 6-week old C57BL/6JJmsSlc mice (20–25 g) were purchased from Shizuoka Laboratory Center (SLC), Inc. Japan. All mice were housed and acclimatized to their new environment for 1 week in a temperature- (23–25 °C) and humidity- (55%–60%) controlled room under a 12 h:12 h light/dark cycle. The mice were divided into four batches of 12 mice for two sets of experiments: 8 h and 24 h Mohs’ paste application experiments. Mice in all groups were provided food and water ad libitum. This study was conducted according to Japanese law concerning the protection and control of animals and the Animal Experimental Guidelines of Yokohama University of Pharmacy (https://www.hamayaku.ac.jp/images/material/56/files/animal_01_2020.pdf). The study protocol was approved by the Animal Ethics Review Committee of our university, and all efforts were taken to minimize animal pain and stress during the study. Six mice per group were assigned for molecular studies, and the remaining six mice were assigned for histopathological studies of angiogenesis and analyses of blood vessel density.

Surgical procedures

All mice underwent unilateral hind limb surgery 24 h before application with Mohs’ paste. Unilateral hind limb surgery was performed following a protocol [26], and the experimental design for the ischemic operation is shown in Fig. 1. Briefly, operative intervention was performed by creating an incision in the skin overlying the middle portion of the right hind limb of each mouse. After ligating the proximal end of the femoral artery, the proximal end of the popliteal artery was ligated. The artery and all side branches were dissected free, and the femoral artery and attached side branches were removed. The overlying skin was sutured using surgical thread. The mice were then used in subsequent experiments as ischemia model mice.

Fig. 1.

Schematic image of the experimental design on ischemic operation in the femoral artery and popliteal artery. Arrows show the sites of ligation and the femoral artery, where the attached side-branches were removed

Measurement of blood flow

Blood flow was measured using a laser Doppler perfusion imaging system (MoorLDI2-2λsim, Moor Instruments Ltd., UK) at a depth of 2 mm from the surface. Mice were maintained at 37 °C using a KN-475 type III animal warming pad (Natsume Seisakusho Co., Ltd, Tokyo, Japan) under isoflurane anesthesia (1%–3%) [27]. In the analysis of blood flow, the background was adjusted to reduce measurement noise, and the ratio of the operated leg (right side) and the control leg (left side; reference) was obtained.

Sample preparation and muscle extraction for ICP-MS analysis

At the end of the experiment, the mice were euthanized and their bilateral hind limb muscles were dissected out. The muscles were frozen using isopentane in liquid nitrogen. Frozen samples were stored at -80 °C until analysis. Intramuscular zinc extraction was performed following modified protocols [28, 29]. Briefly, 50 mg of the sample was accurately weighed into a quartz digestion vessel. Eight milliliters of 69% nitric acid (Ultrapure-100 nitric acid 1.42, Kanto Chemical Co., Inc., Tokyo, Japan) and 1 mL of 30% hydrogen peroxide (Ultrapure-Acid hydrogen peroxide, Kanto Chemical Co., Inc.) were added to each vessel. A cobalt solution (0.1 mg) was also added as an internal standard, and then each vessel was closed. Microwave digestion was conducted by applying a three-step program as follows: 200 W for 3 min twice, and then 3 min at 500 W. After the vessels had cooled down, the digests were transferred into 15 mL tubes and diluted ten-fold with 5% nitric acid.

Quantification of intramuscular zinc in the hind limbs

Total intramuscular zinc in the hind limb was quantified using inductively coupled plasma mass spectrometry (ICP-MS; iCAP Qc ICP-MS, Thermo Fisher Scientific Inc., Waltham, MA, USA) following a previously described protocol [30]. The analyses were conducted under the following conditions: RF power, 1550 W; plasma argon gas flow rate, 14 L/min; carrier argon gas flow rate, 1.0 L/min; auxiliary gas flow rate 0.8 L/min; helium gas flow rate, 4.5 L/min; electric voltage for kinetic energy discrimination, + 3 V. A concentric nebulizer, nickel sampling cone, and nickel skimmer cone were used. The ICP-MS detection masses were set at m/z ratios of 64 (64Sn + ; Zn) and 59 (59Sn + ; Co). We selected an m/z ratio of 64, which is a widely used target ratio for zinc analysis [31]. Samples were diluted ten-fold with 5% nitric acid before subjected to ICP-MS analysis.

Western blot analysis

To understand the mechanism of action of Mohs’ paste in angiogenesis, VEGF-A levels were analyzed by western blot analysis. Samples (n = 3 in each group) containing 10 μg of hind limb muscle proteins were prepared as described in our previous study [32]. In this study, protein concentrations were determined using a Micro BCA Protein Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). In the analyses, 12% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Hercules, CA, USA) and polyvinylidene fluoride (PVDF) membranes were used. Nonspecific binding was blocked using Tris-buffered saline with 0.1% Tween-20 (TBS-T) and 5% bovine serum albumin (Wako Pure Chemical Industries, Ltd. Osaka, Japan), for 1 h at room temperature. The membranes were then washed with 0.1% TBS-T (3 times, 5 min each) and incubated overnight at 4 °C with primary antibodies against VEGF-A (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Immunoreactive bands were detected using a horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Protein bands were visualized using ECL reagents and RX-U Fuji X-ray film (Fuji Film Co., Tokyo, Japan). Images were analyzed using Image J software (freely available from the National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to the level of GAPDH protein in the same membrane.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Comparisons between multiple administration groups and the corresponding control in each experiment were tested using one-way analysis of variance (ANOVA) followed by Dunnett's test. The trend in blood flow levels and the amount of intramuscular zinc, as measured using a laser Doppler perfusion imaging system and ICP-MS, was analyzed using linear regression analysis following our previous study [33]. Statistical analyses were performed using JMP software (version 13.0; SAS Institute, Cary, NC, USA). Differences were considered statistically significant at probability (p) values < 0.05.

Results

Changes in blood flow levels after Mohs’ paste application

A schematic of the experimental design of the ischemic operation is shown in Fig. 1. This model was confirmed to be a moderate ischemic model because the ischemic state decreased to approximately 60% compared to the control group (Table 1). Figure 2 shows representative images of the laser Doppler perfusion imaging system in the right limb pre-operation, as well as the control (0%), 25%, and 50% groups. These images were used to analyze the blood flow levels. The ratios of blood flow levels standardized on the left limb are shown in Table 1. The blood flow level of the control group was recovered 24 h. The blood flow level of the 50% Mohs’ paste application group was significantly lower than that of the 0% group at 8 h (p = 0.0497). At 24 h after application, it was significantly decreased in the 25% (p = 0.0372) and 50% groups (p = 0.0242).

Table 1.

Measurement blood flow levels using a laser Doppler perfusion imaging system

	Time after application
	0 h	2 h	4 h	8 h	24 h
0%	0.58 ± 0.10	0.59 ± 0.02	0.56 ± 0.06	0.72 ± 0.09	0.71 ± 0.05
25%	0.58 ± 0.11	0.65 ± 0.03	0.55 ± 0.08	0.65 ± 0.12	0.60 ± 0.09*
50%	0.63 ± 0.07	0.54 ± 0.01	0.58 ± 0.07	0.60 ± 0.10*	0.59 ± 0.09*

Blood flow levels were measured using a laser Doppler perfusion imaging system and quantified using an attached image analyzer. Data are mean values of the ratios of the right and left legs ± SD of six mice in each group. *p < 0.05, compared with the control

Fig. 2.

Representative laser Doppler perfusion imaging system infrared images from the hind limb ischemia mice at a depth of 2 mm from the surface pre-operation (A), and the control, 25%, and 50% application (B to D) after 24 h. The blue color indicates the part with low blood flow, and the red color indicates the part with high blood flow

Quantification of zinc ion concentrations using ICP-MS

The amount of intramuscular zinc in the hind limb at 8 h and 24 h after Mohs’ paste application was detected using ICP-MS. The quantitative ratio of zinc concentration in standardized left limb muscle was significantly increased in the 50% (p = 0.0474) and 75% (p < 0.0001) groups compared with that of the 0% group at 8 h (Table 2). There was no significant change at 24 h after application in any of the groups.

Table 2.

Measurement of intramuscular zinc concentration using ICP-MS

	Time after application
	8 h	24 h
0%	1.07 ± 0.11	1.18 ± 0.15
50%	5.17 ± 1.21*	2.70 ± 1.07
75%	13.48 ± 5.28**	4.22 ± 3.86

The intramuscular zinc concentration was determined using ICP-MS. Data are mean values, and are the ratios of the right and left legs ± SD of 5 (0% and 50%) or 3 (75%) mice. *p < 0.05, **p < 0.01, compared with the control

Correlation between blood flow and zinc concentration

To understand the dose-dependence trend, we evaluated the relationship between blood flow and zinc concentration using linear regression analysis (Table 3). The results showed a significant effect on blood flow and concentration of zinc in the hind limb-related application dose at 8 h (p = 0.0239 and p = 0.0002, respectively). However, there was no significant relationship between the application dose and the zinc concentration at 24 h.

Table 3.

Coefficients of linear regression analysis

Parameter	Application for 8 h		Application for 24 h
	Coefficient ± SE	p-value	Coefficient ± SE	p-value
Blood flow level
Intercept	0.72 ± 0.03	< .0001	0.70 ± 0.03	< .0001
Application level (× 10−2 per %)	−0.25 ± 0.10	0.0239	−0.25 ± 0.08	0.0094
Zinc concentration
Intercept	0.11 ± 1.28	0.9321	1.09 ± 0.86	0.2346
Application level (× 10−2 per %)	14.39 ± 2.78	0.0002	3.62 ± 1.80	0.0717

Different blood flow levels and zinc concentrations were demonstrated by the laser Doppler system and ICM-MS in the hind limb. Data are presented as the mean coefficient ± standard error (SE)

VEGF-A levels

To understand the mechanisms of Mohs’ paste for angiogenesis, we performed western blot analysis to confirm the VEGF-A protein levels. The results showed that VEGF-A was significantly increased in the 50% and 75% groups at 8 h (p < 0.0001 and p = 0.0014, respectively). No significant change was observed in the expression of VEGF-A in the 50% and 75% application groups at 24 h (Fig. 3A, B).

Fig. 3.

A Results of western blot analysis for VEGF-A in the hind limb muscle of mice administered with Mohs’ paste, B relative quantities. Modifications to the relative protein expression levels of VEGF-A were investigated via western blot analysis. GAPDH served as a loading reference. Relative protein level was expressed as the mean ± SD. **p < 0.01, compared with the control. n = 3 mice in each group

Discussion

Mohs’ paste is used clinically as a hemostatic ointment. Here, we investigated the suppression of angiogenesis using a mouse model of ischemia, which has the ability to recover blood flow within 48 h after ischemia. The present study investigates the intramuscular zinc amount in experimental animals, and provides pharmacological evidence of the therapeutic effects of Mohs’ paste on blood flow levels and the prevention of angiogenesis. A previous study demonstrated that it has a rapid clinical therapeutic effect for hemorrhages induced by disintegrated breast cancer [9]. Suppression of blood flow levels observed in the present study may provide evidence of a hemostatic effect in human cases treated with Mohs’ paste.

Intramuscular zinc concentrations were analyzed using ICP-MS, and more than 50% application groups showed a significant increase after 8 h compared with the control group. Based on these results, we considered that Mohs’ paste permeated to the tissue via the incision after ischemic surgery 8 h after application and increased the intramuscular zinc concentration. However, since blood flow levels showed a decrease at 8 h after 50% application, zinc ions may have a direct effect on the suppression of blood flow levels. On the other hand, the intramuscular zinc level sharply declined to approximately the same level as that of the control after 24 h application, where blood flow even decreased compared to the control. This result revealed that blood flow was suppressed, suggesting that zinc has an indirect effect on the decline in the blood flow levels. The metabolic fate of zinc is excretion as feces. During zinc excess, Zrt- and Irt-like protein 5 (ZIP5) and zinc transporter 2 (ZnT2), which are intestinal zinc transporters, are involved in the metabolism of pancreatic secretions. ZIP5 sequesters zinc from the plasma, while ZnT2 excretes zinc into zymogen granules, both of which are subsequently excreted into the gastrointestinal tract [12, 34–36]. Thus, a decrease in the intramuscular zinc amount after 24 h application suggests its excretion by pancreatic secretion via ZIP5 and ZnT2, and subsequent excretion as feces.

We demonstrated the molecular mechanisms of Mohs’ paste on angiogenesis using western blotting. VEGF-A levels were significantly increased in the 50% and 75% groups after 8 h of application, while there was no significant change after 24 h. VEGF-A is known as the main factor that interacts with VEGF receptors -1 and -2 on the endothelial cell membrane, contributing to angiogenesis in ischemic conditions, and its expression is induced by zinc in animals [20, 37]. The mechanism of zinc-induced VEGF expression involves extracellular zinc-mediated activation of the promiscuous zinc-sensing receptor (ZnR)/G protein-coupled receptor 39 (GPR39), which triggers Gq signaling pathways, activation of phospholipase C (PLC), serine/threonine protein kinase B (Akt), phosphoinositide 3 (PI3), and mitogen-activated protein (MAP) signaling pathways [20]. However, zinc is also essential for the stabilization of endostatin, which is known as an anti-angiogenic activating factor [38]. Zinc binding contributes to the biological functions of endostatin, and has been reported to suppress VEGF receptor-2 [39]. Following the inhibition of VEGF receptor-2 activity, VEGF protein is upregulated. Moreover, it is considered that the release of angiogenic factors associated with the escape from the ischemic state contributed to the increase in VEGF-A levels [40, 41]. This could plausibly explain the increase in VEGF-A levels along with the amount of intramuscular zinc. Further studies are required to determine how increased VEGF-A levels affect hemostasis upon application of Mohs’ paste. In vivo studies have revealed that the application of Mohs’ paste for 24 h decreased not only iNOS messenger RNA expression levels but also CD31-positive cells in the mouse hind limb muscle [9]. Interestingly, our findings showed that the recovery of blood flow continued to be suppressed post 8 h of application, and it was revealed that Mohs’ paste suppressed the recovery of blood flow regardless of the VEGF-A level. It was thus, suggested that Mohs’ paste application may contribute to a reduction in the number of blood vessels and iNOS expression levels despite the elevated levels of the angiogenic factor VEGF-A.

There are limitations in the present study. First, the present study did not evaluate the same concentration of Mohs’ paste between blood flow levels and the analysis of zinc/VEGF-A levels. We considered that this difference could be solved by linear regression analysis; however, further experiments are required to investigate the effect of different concentrations that has not been demonstrated in the present study. Second, the present study did not evaluate major angiogenesis-related markers, such as endostatin, angiogenin, and hypoxia-inducible factor (HIF) -1. Moreover, the lack of data on hemostatic observation is a limitation of the present study. Therefore, this is a preliminary study on angiogenesis, and further studies are required to determine whether the application of Mohs’ paste affects ischemia-induced angiogenesis.

Conclusion

Mohs’ paste showed that blood flow recovery was suppressed in a zinc concentration-dependent manner at 8 h after application. Moreover, the mechanism of blood flow suppression was suggested to be independent of VEGF-A levels and might also suppress angiogenesis. Our findings support previous studies that identified a decrease in CD31-positive cells, and Mohs’ paste was characterized by two different pharmacological effects being exhibited at 8 h and 24 h post-application. The present study provides evidence of an anti-angiogenic effect and can be appropriate for situations that require hemostasis in the clinical setting.

Abbreviations

ICP-MS

Inductively coupled plasma mass spectrometry

VEGF

Vascular endothelial growth factor

iNOS

Inducible nitric oxide synthase

ZIP

Zrt- and Irt-like protein

ZFP

Zinc finger proteins

ZnT

Zinc transporter

ZnR

Zinc-sensing receptor

ADP

Adenosine diphosphate

GPR39

G protein-coupled receptor 39

PLC

Phospholipase C

PI3

Phosphoinositide 3

MAP

Mitogen-activated protein

HIF

Hypoxia-inducible factor

Data Availability

The data supporting the findings of this study are available from the corresponding author, [NI], upon reasonable request.

Code availability

All source codes are available from the corresponding author, [NI], upon reasonable request.

Declarations

Conflict of interest

The authors declare no conflicts of interest.