Published online Nov 19, 2024.
https://doi.org/10.4142/jvs.24163
Medication effects on pulmonary thromboembolism in mice intravenously transplanted with canine adipose tissue-derived mesenchymal stem cells
Abstract
Importance
The intravenous administration of adipose tissue-derived mesenchymal stem cells (AdMSCs) in veterinary medicine is a promising regenerative therapy, but it can lead to severe complications, including pulmonary thromboembolism (PTE).
Objective
As part of an ongoing study, this study examined the impact of medications, such as heparin, aspirin, and sodium nitroprusside (SNP), on the factors linked to PTE after an intravenous injection of canine mesenchymal stem cell into experimental animals.
Methods
Fluorescently labeled canine AdMSCs were administered intravenously into the tail veins of five-week-old male BALB/c hairless mice. This study compared the survival rates, biodistribution, platelet counts, D-dimer levels, and histological examination results among the drug treatment experimental and the control groups.
Results
The final survival rates in the SNP, control aspirin, and heparin groups were 25%, 33%, 50%, and 100%, respectively. Ex vivo imaging confirmed fluorescence exclusively in the lungs of all subjects who died during the injection, with no fluorescence detected in the other organs. On the other hand, in the heparin experimental group, the surviving individuals exhibited fluorescence in the lungs and the liver on day one. Histological biopsies revealed PTE in all deceased individuals within the medication experimental groups (p = 0.029).
Conclusions and Relevance
Heparin was highly effective, with no PTE-related deaths observed when used alongside cell injections. Aspirin revealed moderate effectiveness, surpassing the control group. On the other hand, the efficacy of SNP was inferior to that of the other two drugs.
INTRODUCTION
Stem cell therapy in veterinary medicine has attracted increasing attention as a treatment in various fields. Among the different types of stem cells, adult stem cells, particularly adipose tissue-derived mesenchymal stem cells (AdMSCs), are a widely used option in veterinary and human medicine because of their ethical advantages, low risk of infections, reduced immune rejection responses, and the ease of collection and cultivation [1, 2]. Regarding the injection method of these stem cells, direct administration to the target organ carries potential side effects and risks, including bleeding and tissue damage [3]. Hence, the intravenous injection method is recommended for systemic stem cell administration because of its non-invasive nature and high accessibility compared to other methods [3].
Nevertheless, potential complications associated with intravenous injection of mesenchymal stem cells (MSCs) exist, including tumor formation, failure of differentiation into target organs, and pulmonary embolism (PE) during the injection process [2, 4, 5, 6, 7]. The failure of lung passage before reaching the target organ and subsequent PE have been highlighted as adverse events in most studies [2, 4, 5, 6, 7].
The mechanism of such pulmonary thromboembolism (PTE) induction can be divided into causes by the coagulation mechanism involving tissue factor expression and instant-blood mediated inflammatory reaction (IBMIR) [8, 9, 10] and the cause of physical trapping owing to the size of MSCs, which are larger than pulmonary capillaries [4, 11, 12, 13]. Previous in vitro experiments involved measurements of the MSC sizes, followed by subsequent in vivo experiments that compared the outcomes while accounting for various factors associated with lung passage and the induction of embolism [14].
This study extends previous research by investigating the effects of medications on pulmonary passage and embolism-inducing factors. In particular, drug test groups receiving concurrent administration of drugs, such as heparin, aspirin, and sodium nitroprusside (SNP), were compared and evaluated against a control group injected only with stem cells. The clinical symptoms were observed. The survival rates were evaluated. Spectral Ami HT biodistribution analysis was conducted, and the platelet and D-dimer values were measured through blood tests. The presence of PTE was confirmed through a lung biopsy following an injection of canine AdMSCs into the mouse tail vein, as reported elsewhere [14].
METHODS
Preparation of canine AdMSCs
The process for preparing canine AdMSCs was consistent with the methodology used in the previous study [14]. Canine AdMSCs, ranging from 5 × 105 to 5 × 106 cells, were cryopreserved using 1 mL of cell banker (Cell Banker 1, Nippon Zenyaku Kogyo Co., Ltd., Japan). All AdMSCs used in the experiment were within three passages. The cryopreserved AdMSCs were thawed using a constant-temperature water bath oscillator (Taitec Corporation, Japan) set at 37°C. The MSCs were then washed 2–3 times with full medium (complete stromal media), comprising Dulbecco’s Modified Eagle Medium/nutrient mixture F-12 (DMEM/F12, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Tissue Culture Biologicals, USA) and 1% antibiotic-antimycotic (AA, Thermo Fisher Scientific). After each wash, the solution was centrifuged at 1,500 rpm for 5 min, and the supernatant was carefully aspirated. The remaining MSCs were then resuspended in full medium.
Fluorescent labeling of canine AdMSCs
Canine adipose tissue-derived mesenchymal stem cells (cAdMSCs) were resuspended in phosphate-buffered saline (PBS, Biowest, France) at 1 × 106 cells/mL. Subsequently, 20 μL of CytoFlamma 749 cell membrane solution (Bioacts, Incheon, Korea) per mL was added to the suspension and mixed thoroughly by pipetting. The labeled MSCs were incubated at 37°C for 30 min, with agitation every 10 min. After incubation, the labeled MSCs were centrifuged at 1,300 rpm and 37°C for 3 min.
Cell injection in mice
Five-week-old male BALB/c hairless mice were sourced from DBL (Korea). After a one-week acclimation period to the environmental conditions, the mice were given access to water and food pellets ad libitum. All procedures involving animals adhered to Konkuk University’s guidelines for animal use and were approved by the Institutional Animal Care and Use Committee (IACUC number KU17182-1).
Consistent with previous research, the mice were immobilized in a restraint frame for cell injection, and their tails were placed in warm water for approximately 5 min to facilitate blood vessel dilation [14]. After disinfection with 70% alcohol, fluorescently labeled cAdMSCs were injected into the tail vein using a 0.5 mL insulin syringe (31 G) (Ultra-Fine II short needle, Becton-Dickinson and Company, USA) containing a 200 μL PBS suspension at an injection rate of 200 μL per minute. Bleeding at the injection site was managed with gauze. In the control group of six animals, 2 × 106 cAdMSCs were injected as a single bolus in 200 μL of PBS suspension [14].
Administration of medications (anticoagulant [heparin], antiplatelet [aspirin], and vasodilator [SNP])
Before administration, heparin (Heparin Sodium Injection, JW Pharmaceutical, Korea) was diluted in the suspension at 100 U/kg and co-administered with the stem cells via the tail vein. In particular, 2 × 106 cells were diluted in 200 μL of a PBS suspension with 100 U/kg heparin and injected at an infusion rate of 200 μL/1 min.
Before the MSC injection, aspirin (Rhonal, KunWha Pharmaceutical Co., Korea) at a dose of 43 mg/kg was administered orally twice daily for seven days. Subsequently, 2 × 106 cells were diluted in 200 μL of a PBS suspension and injected at an infusion rate of 200 μL/1 min using the same procedure.
A 1 μL volume of SNP (Nitropress, Hospira, Inc., USA) at a concentration of 25 mg/mL was diluted in 200 μL of normal saline (0.9% NS, JW Pharmaceutical) and administered intravenously 5 min before the stem cell injection. Subsequently, 2 × 106 cells were diluted in 200 μL PBS suspension and injected at an infusion rate of 200 μL/1 min using the same procedure.
The heparin group consisted of seven animals, while the aspirin and SNP groups contained eight animals each.
In vivo and ex vivo fluorescence imaging for biodistribution analysis
After transplantation, the mice were placed under anesthesia using 4% isoflurane (IFRAN LIQ, Hana Pharm, Co., Korea) in the light-tight specimen chamber of the Spectral Advanced molecular imaging (Ami) HTX (Spectral Instruments Imaging, USA), as reported elsewhere [14]. Anesthesia was administered prior to imaging. On the other hand, in cases where mice died after a cell injection, they were photographed in the deceased state. In vivo imaging was performed on days one, two, three, and seven, while ex vivo imaging was conducted only on the first and last days. All images were captured using a charged-coupled device camera at excitation and emission wavelengths of 756 nm and 785 nm, respectively. For in vivo scans, the regions of interest (ROI) in the pulmonary region of the images were selected. In the previous study, in vitro samples were imaged in 100 μL of PBS in a 96-well plate (SPL Life Sciences, Korea), and ROI values were obtained based on the concentration [14]. In the case of ex vivo imaging, the organs were excised from the mice, and the ROI values were measured to identify the distribution of injected cells within the organs. The fluorescence signals of the ROI were quantified in photon counts per second (p/s) using Spectral Instruments Imaging software.
Post-injection monitoring related to PTE
After the cell injection, the experimental animals were monitored closely for the clinical symptoms and overall survival. Blood samples were obtained from the femoral vein to assess the complete blood count (CBC) values, particularly platelet counts and D-dimer levels, on the first day after the cell injection. In cases where animals did not survive, blood samples were collected by exsanguination from the caudal vena cava for histological analysis. The CBC values were determined using the URIT-2900 VET Plus hematology analyzer (URIT Medical Electronics, China). The D-dimer levels in mice were assessed by isolating plasma and using the Mouse D-Dimer (D2D) ELISA Kit (MBS269348, MyBioSource.com, USA), according to the manufacturer’s instructions.
Histology
In instances where the experimental animals expired during the injection or monitoring period, lung tissues were promptly examined postmortem via lung biopsies to detect complications such as PE. Surviving subjects were euthanized by intravenous administration of 1–2 mEq of potassium-chloride (KCl, potassium chloride-40 injection, Dai Han Pharm, Korea) under 4% isoflurane (IFRAN LIQ, Hana Pharm, Co.) anesthesia after imaging on the final day of the experiment (day seven). Lung fixation was carried out using the fixed-volume fixation method [15]. The thorax was opened, and the caudal vena cava was incised to exsanguinate. A cannula, using a 24 G IV catheter (BD Angiocath Plus, Becton-Dickinson and Company, USA), was inserted into the trachea, and the ligature was secured using Dafilon 4-0 (Dafilon 4/0, B. Braun Surgical, Spain). The lungs were fixed by continuous infusion of 10% formalin fixative through the cannula using an infusion pump (12 mL/h; 5 min). Embedding was carried out within 24 h post-fixation, and blocks were prepared. Lung tissues were sectioned at a depth of 5 μm at 200, 400, 600, and 800 μm in the dorsal-ventral plane and stained with hematoxylin and eosin. Within the lung tissue, the endothelium surrounding the blood vessels near the bronchi was observed. Areas where blood clots composed of white blood cells, red blood cells, and fibrin were adhered were identified and imaged at × 100 and × 200 magnification using an optical microscope (Olympus PROVIS AX70, Japan), Nikon DS-Ri2 camera (Japan), and NIS-Elements BR 4.50.00 software (Tokyo, Japan). The number of vessels exhibiting thromboembolism was graded as 0 (normal), 1 (low), 2 (moderate), and 3 (high).
Statistical analysis
All digitized data were analyzed using software (v29.0, SPSS Statistics, IBM, USA). The significance of the control group and the first day in vivo fluorescence imaging ROI values, platelet counts, plasma D-dimer levels, and PTE grades in the three experimental groups were evaluated. Owing to the small sample size, a nonparametric statistical method that does not require assumptions of normality and the homogeneity of the variance was used. The initial normality assessments (Kolmogorov–Smirnov and Shapiro–Wilk test) showed that the data did not conform to normality assumptions. The Kruskal–Wallis test was used to assess the significance among all experimental groups; p values < 0.05 were considered significant. A Fisher’s exact test was used to evaluate the significance of the survival rate between the two experimental groups because the cells with a frequency of less than five accounted for more than 20% of the total. All experimental data are presented as mean ± SD.
RESULTS
Post-injection clinical manifestation and survival rate
All the experimental animals that died in the present study experienced immediate death upon the injection or within minutes of the injection. Some individuals exhibited acute adverse symptoms such as convulsions, shivering, dyspnea, and tetraplegia. The final survival rates in the SNP, control aspirin, and heparin groups were 25%, 33.33%, 50%, and 100%, respectively. Significant differences existed between the control – heparin group (p = 0.021) and heparin – SNP group (p = 0.007). Table 1 lists the survival rates of all experimental groups.
Table 1
Survival rates of the experimental animals on day seven
Post-injection blood tests (platelet, D-dimer)
The mean values of the platelets were observed in the following order: heparin, aspirin, control, and SNP group (p = 0.074). In all experimental groups using drugs, the average D-dimer values were lower than those of the control group, and the average values were higher in the following order: aspirin, SNP, and heparin group (p = 0.86). Fig. 1 show the box whisker plot graphs representing values for platelets and D-dimer.
Fig. 1
Box whisker plot graphs of the platelet counts and D-dimer levels. (A) Box whisker plot graphs of platelet counts. (B) Boxplot graphs of D-dimer levels. Each panel presents descriptive statistics for the platelet counts and D-dimer levels, including the maximum, upper quartile, interquartile range, median, lower quartile, and minimum values.
SNP, sodium nitroprusside.
In vivo and ex vivo fluorescence imaging for biodistribution analysis
Previous in vitro fluorescence imaging experiments have shown that the fluorescence expression increases as the cell concentration increases [14]. The in vivo ROI levels of the day one data showed significant differences between the control and the medication experimental groups (p = 0.022). Fig. 2A shows in vivo fluorescence photographs of the control group taken on each experimental date. Although not included in the text, the remaining drug experimental groups were photographed in the same manner. Fig. 2B presents all the numerical ROI data and mean values in a graph.
Fig. 2
In vivo fluorescence imaging. (A) In vivo fluorescence imaging over time in the control experimental group. The bar of the pseudo-color image indicates the ranges of light intensity levels. (B) Time course graphs of in vivo fluorescence ROI values. All ROI values of the experimental groups by dates are shown in the figures, and the average values are indicated by a line. The ROI values of the front and back measured in one object are indicated, respectively. This means there are two measurements per individual (front/back). The measured values per object are all expressed in the same figure. In other words, the front and back are marked as one figure without any difference.
ROI, regions of interest; SNP, sodium nitroprusside.
For ex vivo imaging, one individual from each experimental group was selected, euthanized, and their organs were extracted. Among the individuals who died immediately upon injection, fluorescence was only observed in the lungs; no fluorescence was detected in organs other than the lungs. In the surviving individuals treated with medication, the fluorescence was observed in the liver and lungs of the heparin experimental group on day one, and fluorescence was detected in the liver, spleen, and lungs of all experimental groups on day seven. The ex vivo ROI data were visualized as images and graphs in Fig. 3.
Fig. 3
Ex vivo fluorescence imaging. (A) Ex vivo fluorescence imaging. The color bar in the pseudo-color image indicates ranges of light intensity levels. In particular, the numbers 1, 2, 3, 4, 5, and 6 correspond to the heart, lung, liver, spleen, kidneys, and intestines, respectively. The negative control is an object injected with saline instead of mesenchymal stem cells. (B) Bar graphs of ex vivo fluorescence regions of interest values. The “live/dead” column denotes the status (survival) of the experimental animal at the time of the measurement.
SNP, sodium nitroprusside.
Histology
The histology results showed that all deceased individuals in the medication experimental groups exhibited PTE upon biopsy (10/10). On the other hand, no PTE was detected in the surviving individuals from the medication experimental groups (0/13). The individuals with confirmed PTE on biopsy showed fibrin pigmentation within pulmonary vessels and plaque formation. The average PTE grade was observed in the following order: SNP group (2.13 ± 1.36, n = 8), aspirin group (1.38 ± 1.51, n = 8), control group (0.83 ± 1.17, n = 6), and heparin group (0.00 ± 0.00, n = 7). The differences between the groups were significant (p = 0.029). A pairwise comparison indicated significant differences between the heparin and SNP experimental groups (p = 0.018). Furthermore, the ratio of confirmed PTE was observed in the order of SNP, control/aspirin, and heparin groups. Fig. 4 compares the photomicrographs showing normal findings in a biopsy and micrographs with confirmed PTE. Fig. 5 presents a graphic display of the PTE grade and ratio.
Fig. 4
Representative photomicrographs of lung tissue from experimental animals showing normal or PTE findings. (A) Normal (scale bar = 100 µm). (B) Normal (scale bar = 100 µm). (C) Normal (scale bar = 50 µm). (D) PTE (scale bar = 100 µm). (E) PTE (scale bar = 50 µm). (F) PTE (scale bar = 50 µm). The PTE samples show specific features, including prominent endothelium, minimal plaque formation, and amorphous round fibrin-like materials within the blood vessels (arrows). (A-F) hematoxylin and eosin staining.
PTE, pulmonary thromboembolism.
Fig. 5
Graphs of the PTE grades and ratio. The classification criteria for PTE grades are as follows: grade 0 (normal/thromboembolism confirmed in < 10% of the vessels); grade 1 (low/thromboembolism confirmed in 10%–30% of the vessels); grade 2 (mid/thromboembolism confirmed in 30%–60% of the vessels); grade 3 (high/thromboembolism confirmed in > 60% of the vessels).
PTE, pulmonary thromboembolism; SNP, sodium nitroprusside.
DISCUSSION
This study compared the effects of drugs related to PTE, a potential adverse effect of intravenous stem cell administration. According to the review paper by Louise Coppin et al. [16] in 2019, which summarized the thrombogenic risk associated with intravascular injection of MSCs, controlling procoagulation, the primary mechanism behind PTE, is crucial for enhancing the therapeutic effect. Hence, modulating procoagulant activity (PCA) is essential. Moreover, several papers have been published showing anticoagulants (such as heparin and bivalirudin) during an intravascular injection of MSCs [16]. In vitro and in vivo studies have shown that the PCA of MSCs, which typically occurs upon exposure to blood, can be inhibited or restricted by anticoagulants, including heparin [16]. Heparin, in particular, is used widely and has been proven effective in various studies in thrombosis research and studies investigating the efficacy of MSCs, even in the veterinary field [16, 17, 18]. The experimental design in the present study was based on Fischer et al. [12], and the heparin concentration was adjusted according to the body weight. The results revealed a 100% survival rate in the subjects injected with heparin, demonstrating excellent efficacy. Furthermore, the average platelet count in the blood tests was highest in the experimental groups receiving heparin, while the D-dimer level was the lowest. These findings suggest that heparin effectively controlled the PCA of MSCs.
Aspirin, or acetylsalicylic acid, is a well-known antithrombotic agent that exerts its effects by inhibiting platelet activation and aggregation, reducing thrombin production, impairing neutrophil extracellular trap formation, and promoting the formation of soluble fibrin networks [19]. Aspirin has been studied widely for its preventive effects against venous thromboembolism (VTE) and PE, including deep vein thrombosis [20, 21, 22]. Aspirin is commonly used as a prophylaxis for VTE in human medicine, particularly in arthroplasty procedures [23, 24, 25]. The effective dose of aspirin for VTE prevention in humans varies, but many studies, including the American Academy of Orthopedic Surgeons (AAOS) guidelines, suggest a dose of 325 mg twice daily [24, 25, 26]. In the present experiment, mice were administered an aspirin dose equivalent to 43 mg/kg based on a body surface area dose conversion method [27], corresponding to a dose of 325 mg/day in humans. The mice received oral aspirin twice daily for seven days before the MSC injection. The results showed a higher survival rate than the control group without drug use but lower than the group administered heparin, which exhibited a 100% survival rate. Hence, while many studies on heparin directly modulated the PCA of MSCs, few studies have examined the combination of MSCs intravascular administration and antithrombotic drugs such as aspirin. Moreover, in human medicine, the exact dose, usage, and duration of aspirin as prophylaxis have not been established [28]. Therefore, the concentration or duration of aspirin in the present experiment, particularly in combination with MSCs, may have been insufficient in mice. Nevertheless, in the present experiment, the average platelet count was lower than that of the negative control group, which was injected with normal saline (0.9% NS, JW Pharmaceutical) (169.33 ± 39.51 109/L, n = 3), proving its efficacy as an antiplatelet to some extent. On the other hand, the precise impact of aspirin on PCA when administered in combination with MSCs has not been verified. As mentioned earlier, studies on the combined administration of aspirin with MSCs have not been conducted, and further research in this area is anticipated.
SNP is a vasodilator commonly used as a hypotensive agent in clinical practice [29, 30, 31]. It acts by releasing nitric oxide (NO), which increases the levels of cyclic guanosine monophosphate (cGMP), activates cGMP-dependent kinase, and ultimately inhibits smooth muscle contraction by affecting the intracellular calcium ion concentration [29, 30, 31]. In addition, the release of NO and increased cGMP inhibit platelet aggregation and clot formation [29, 30, 31]. Several studies have explored the use of SNP in conjunction with intravascular injection of MSCs [13, 32]. Gao et al. [32] reported a 15% reduction in the number of MSCs identified in the lungs when SNP was administered before intra-arterial or intravenous injection of rat bone marrow-derived MSCs, while more cells reached other organs such as the liver, kidney, and bone.
Similarly, Schrepfer et al. [13] used SNP in an intravenous injection experiment of bone marrow-derived MSCs in mice. They reported more cells passing through the lung barrier than the control group. In the present experiment, however, the survival rate was lower, and the average PTE grade and PTE ratio on biopsy were higher than the control group injected with 2 × 106 cells without medications. Various experimental errors may have occurred in the SNP experimental group. In the present study, the use of SNP was based on a mouse experimental design paper. The average MSCs may be similar because this experiment used MSCs derived from dog adipose tissue, but the effectiveness of SNP may have been compromised considering the deviation between animals.
Nevertheless, even with these potential errors, the use of anticoagulants and antiplatelet agents, which directly influence blood coagulation, yielded better results than SNP. These findings suggest that while SNP may have some impact on blood coagulation, its main action is the dilation of blood vessels. The results suggest that directly targeting blood coagulation is a more effective strategy than solely focusing on expanding the mechanical capillary bed. In particular, the superior efficacy of heparin highlights that the IBMIR triggered by MSC PCA is the main mechanism contributing to thromboembolism rather than purely mechanical causes.
In conclusion, this study examined the effects of different medications on the occurrence of PTE in an animal model after the intravenous injection of canine adipose tissue-derived MSCs. Heparin, an anticoagulant, showed the highest efficacy, while aspirin, an antiplatelet agent, showed lower efficacy than the control group. In contrast, the vasodilator SNP produced worse results than the control group. These findings highlight the importance of targeting the coagulation mechanism to reduce the occurrence of PTE associated with intravenous stem cell injections. The use of heparin and aspirin can be effective strategies in mitigating this major side effect.
Nevertheless, research data suggests the lung passage effect of SNP, a vasodilator. The effect of SNP, which contributes to the mechanical PTE-inducing mechanism, can be revealed through a more detailed experimental design that accounts for interspecies differences. Further research will be needed.
This study contributes to the existing knowledge of intravenous stem cell treatments in veterinary medicine and provides valuable insights for developing standardized protocols to enhance treatment safety.
Conflict of Interest:The authors declare no conflicts of interest.
Author Contributions:
Conceptualization: Kwon J, Yoon HY.
Data curation: Kwon J, Kim MY.
Formal analysis: Kwon J, Kim MY.
Investigation: Kwon J.
Methodology: Kwon J.
Project administration: Lee JI, Kim W, Hyun JE, Yoon HY.
Resources: Kwon J.
Software: Kwon J.
Supervision: Yoon HY.
Validation: Kwon J, Yoon HY.
Visualization: Kwon J, Yoon HY.
Writing - original draft: Kwon J.
Writing - review & editing: Kwon J, Yoon HY.
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