Abstract
Objective
Cell therapy is an exciting area of investigation for repair of injured myocardial tissue. Platelet-rich plasma (PRP) is an autologous fractionation of whole blood containing high concentrations of growth factors including vascular endothelial growth factor and insulin-like growth factor, among many others. PRP has been shown to safely and effectively enhance healing of musculoskeletal tissue primarily by reparative cell signaling. Despite a growing body of evidence on PRP’s safety and efficacy, limited studies have been performed using PRP in cardiovascular tissues. Utilizing a murine myocardial permanent ligation and ischemia/reperfusion model, this study sought to determine whether RevaTen PRP (Menlo Park, CA, USA), a proprietary formulation of PRP, improves cardiac function as measured by left ventricular ejection fraction (LVEF).
Methods
Via thoracotomy, the left anterior descending arteries (LAD) of 28 mice were occluded by suture either permanently or for 45 min to induce ischemic injury and then reperfused. Mice undergoing permanent ligation had intramyocardial injections of either RevaTen PRP ( n =5) or phosphate-buffered saline (PBS; n =4). Magnetic resonance (MR) imaging was performed to calculate LVEF at 7 days. Mice undergoing ischemia and reperfusion had intramyocardial injections of either PRP ( n =10) or PBS ( n =9) and underwent MR imaging to calculate LVEF at 21 days. Hearts were harvested for histologic examination following imaging.
Results
Compared with PBS controls, RevaTen PRP-treated animals that underwent LAD ligation had a 38% higher LVEF 7 days after injury (PRP=36.1±6.1%; PBS=26.4±3.6%, P =.027). Compared with PBS controls, PRP-treated animals who underwent ischemia–reperfusion of the LAD had a 28% higher LVEF 21 days after injury (PRP=37.6±4.8%, control=29.3±9.7%, P =.038). Histologic analysis suggested the presence of more scar tissue in the control group compared to the PRP-treated animals.
Conclusion
MR imaging demonstrated a positive effect of RevaTen PRP on left ventricular function in both a ligation and ischemia–reperfusion murine model. Our results suggest RevaTen PRP should be investigated further as a potential point-of-care biologic treatment following myocardial injury.
1
Introduction
Despite advances in prevention, pharmacological intervention, and medical procedures, myocardial infarction remains the leading cause of death in industrialized nations . Thus, investigators continue to search for potential biologic therapies to treat damaged myocardial tissue. Although stem cell therapies currently represent an exciting area of research, challenges include cell choice, mode of harvesting and processing, and cell survival. Platelet-rich plasma (PRP) is a form of platelet cell therapy that has emerged as an interesting biologic tool in regenerative medicine. Its benefits include point-of-care availability, simple autologous preparation, and no rejection risks. PRP is a fractionation of autologous whole blood that contains significant quantities of a variety of growth factors including, but not limited to, vascular endothelial growth factor, transforming growth factor-beta (TGF-β), insulin-like growth factor, and platelet-derived growth factor . PRP can be considered a form of autologous cytokine therapy.
PRP has been evaluated as a primary treatment or in conjunction with surgery in a variety of tissues. PRP has been shown to improve the biomechanical properties of tendons, enhance their vascularity, and induce migration of reparative cells to a site of injection . There is also significant evidence supporting the use of PRP in chronic, severe lateral epicondylitis . Other investigations have studied its use in the treatment of rotator cuff , Achilles tendon , and patellar tendon injuries. However, only limited work has been performed evaluating the potential benefits of PRP on cardiovascular tissues. Preclinical studies have been limited to improvement of perfusion in a murine hind limb ischemia model and modulation of post-myocardial infarction remodeling in a rodent model . One clinical study using PRP in patients with chronic angina demonstrated its excellent safety profile . Although PRP has been evaluated and used in a variety of conditions, the most clinically relevant cardiovascular applications have yet to be determined.
Notably, not all PRP is the same. Some forms of PRP contain only concentrated platelets. Others also contain increased white blood cells compared to baseline whole blood. Ex vivo activation of PRP with thrombin and/or calcium produces a gel that can be applied to a surgical site. In vivo activation of PRP via direct contact with collagen-containing tissue is a preferred form of PRP because it results in slower growth factor release at the desired anatomic site . In light of this, Mishra et al. (in press) have proposed a classification system that describes different formulations of PRP and provides a framework for comparing how these different types of PRP function. RevaTen PRP is a proprietary formulation of unactivated PRP containing both highly concentrated platelets and white blood cells. In this investigation, we specifically sought to evaluate how in vivo application of RevaTen PRP influences left ventricular cardiac function after myocardial injury as measured by magnetic resonance imaging using a murine model.
2
Methods and materials
2.1
Animal model
Six- to 8-week-old female NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice (Jackson Laboratory, Bar Harbor, ME, USA) were housed at the Stanford University animal care facility under standard temperature, humidity, and timed-lighting conditions, and provided mouse chow and water ad libitum. All animals were handled in compliance with the National Research Council’s guidelines for the care and use of laboratory animals. A total of 28 mice were selected for two experiments. In both models, human PRP was delivered via intramyocardial injection into severe combined immunodeficient (SCID) mice. With the use of well-established ischemic/reperfusion injury and myocardial infarction models, SCID mice were selected to preclude an immunologic response to the human PRP. SCID mice have impaired VDJ recombination and, consequently, an inability to make T cells, B cells, or components of the complement system; however, they have a functional innate immune system (natural killer cells, macrophages, neutrophils). This murine strain is routinely used in vivo to study the behavior of human cells (humanised mice), where human genes are expressed by mouse cells or transferred human tissue. This approach has been exploited in cancer biology, autoimmunity, allergy, infections, and transplantation research . In the first experiment, nine mice were randomly assigned to experimental (PRP-treated, n =5) or control (PBS-treated, n =4) groups in a permanent ligation model. In the second experiment, 19 mice were randomly assigned to experimental (PRP-treated, n =10) or control (PBS-treated, n =9) groups in an ischemia–reperfusion model.
2.2
Human platelet-rich plasma
RevaTen platelet-rich plasma was prepared from whole human blood from the same donor using a proprietary separation device and process (BioParadox, Menlo Park, CA, USA). After preparation, PRP was buffered to physiologic pH using 8.4% sodium bicarbonate and delivered to the myocardium without exogenous activation. The platelet and white blood cell counts were calculated for each trial before and after the PRP was prepared.
2.3
In vivo myocardial permanent ligation model
NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice were anesthetized and maintained with 3% isoflurane, intubated, and placed on a rodent ventilator. Permanent left anterior descending artery ligation was performed via a left lateral thoracotomy. Fifty microliters of human PRP (experimental group, n =5) or 50 μl of PBS (controls, n =4) was divided into two directly visualized intramyocardial injections in the ischemic region via a 27-gauge needle. The injections were delivered 5 min after artery ligation. The animals were sacrificed on Postoperative Day (POD) 7.
2.4
In vivo myocardial ischemia–reperfusion model
NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice were anesthetized using the same protocol described in the ligation model. Ischemia and reperfusion of the left anterior descending artery were performed via a left lateral thoracotomy and an 8-0 Ethilon suture. The presence of ischemic myocardium confirmed adequate occlusion. After an occlusion time of 45 min, reperfusion of the LAD was allowed for 15 min. Fifty microliters of human PRP (experimental group, n =10) or 50 μl of PBS (controls, n =9) was subsequently divided into two intramyocardial injections in the ischemic region via a 27-gauge needle. The injections were delivered 5 min after artery ligation. Animals were sacrificed on POD 21.
2.5
Myocardial function assessment
Mice were anesthetized with 2% isoflurane with 1 l/min oxygen and placed in the supine position. A small animal ECG and respiratory gating system (Small Animal Instruments, Stony Brook, NY, USA) were applied to acquire images at the end of each QRS and end respiration. Magnetic resonance images were performed on a Signa 3.0 T Excite HD scanner (GE Health Systems, Milwaukee, WI, USA) with a customized small animal surface coil.
Gated gradient-echo fast spoiled GRASS (FSPGR) sequences were used to acquire sequential short-axis slices spaced 1 mm apart from apex to base of the mouse heart. For each sequence, 20 cine frames encompassing one cardiac cycle were obtained with the following sequence parameters: echo time=4.6 ms, number of excitations=2, field of view=50×50 mm, matrix=256×256, flip angle=60°.
A contouring program, Fujin Plus 08 version 3 (Tokyo, Japan), was used to trace the endocardial border of the LV myocardium for each slice of the heart over the entire cardiac cycle in order to determine ejection fraction (EF). This technique has previously been used to evaluate LV function after injection of embryonic stem cells to treat acute myocardial infarction . Following imaging, animals were euthanized and hearts harvested and processed for histology (including hematoxylin and eosin) and trichrome staining.
2
Methods and materials
2.1
Animal model
Six- to 8-week-old female NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice (Jackson Laboratory, Bar Harbor, ME, USA) were housed at the Stanford University animal care facility under standard temperature, humidity, and timed-lighting conditions, and provided mouse chow and water ad libitum. All animals were handled in compliance with the National Research Council’s guidelines for the care and use of laboratory animals. A total of 28 mice were selected for two experiments. In both models, human PRP was delivered via intramyocardial injection into severe combined immunodeficient (SCID) mice. With the use of well-established ischemic/reperfusion injury and myocardial infarction models, SCID mice were selected to preclude an immunologic response to the human PRP. SCID mice have impaired VDJ recombination and, consequently, an inability to make T cells, B cells, or components of the complement system; however, they have a functional innate immune system (natural killer cells, macrophages, neutrophils). This murine strain is routinely used in vivo to study the behavior of human cells (humanised mice), where human genes are expressed by mouse cells or transferred human tissue. This approach has been exploited in cancer biology, autoimmunity, allergy, infections, and transplantation research . In the first experiment, nine mice were randomly assigned to experimental (PRP-treated, n =5) or control (PBS-treated, n =4) groups in a permanent ligation model. In the second experiment, 19 mice were randomly assigned to experimental (PRP-treated, n =10) or control (PBS-treated, n =9) groups in an ischemia–reperfusion model.
2.2
Human platelet-rich plasma
RevaTen platelet-rich plasma was prepared from whole human blood from the same donor using a proprietary separation device and process (BioParadox, Menlo Park, CA, USA). After preparation, PRP was buffered to physiologic pH using 8.4% sodium bicarbonate and delivered to the myocardium without exogenous activation. The platelet and white blood cell counts were calculated for each trial before and after the PRP was prepared.
2.3
In vivo myocardial permanent ligation model
NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice were anesthetized and maintained with 3% isoflurane, intubated, and placed on a rodent ventilator. Permanent left anterior descending artery ligation was performed via a left lateral thoracotomy. Fifty microliters of human PRP (experimental group, n =5) or 50 μl of PBS (controls, n =4) was divided into two directly visualized intramyocardial injections in the ischemic region via a 27-gauge needle. The injections were delivered 5 min after artery ligation. The animals were sacrificed on Postoperative Day (POD) 7.
2.4
In vivo myocardial ischemia–reperfusion model
NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice were anesthetized using the same protocol described in the ligation model. Ischemia and reperfusion of the left anterior descending artery were performed via a left lateral thoracotomy and an 8-0 Ethilon suture. The presence of ischemic myocardium confirmed adequate occlusion. After an occlusion time of 45 min, reperfusion of the LAD was allowed for 15 min. Fifty microliters of human PRP (experimental group, n =10) or 50 μl of PBS (controls, n =9) was subsequently divided into two intramyocardial injections in the ischemic region via a 27-gauge needle. The injections were delivered 5 min after artery ligation. Animals were sacrificed on POD 21.
2.5
Myocardial function assessment
Mice were anesthetized with 2% isoflurane with 1 l/min oxygen and placed in the supine position. A small animal ECG and respiratory gating system (Small Animal Instruments, Stony Brook, NY, USA) were applied to acquire images at the end of each QRS and end respiration. Magnetic resonance images were performed on a Signa 3.0 T Excite HD scanner (GE Health Systems, Milwaukee, WI, USA) with a customized small animal surface coil.
Gated gradient-echo fast spoiled GRASS (FSPGR) sequences were used to acquire sequential short-axis slices spaced 1 mm apart from apex to base of the mouse heart. For each sequence, 20 cine frames encompassing one cardiac cycle were obtained with the following sequence parameters: echo time=4.6 ms, number of excitations=2, field of view=50×50 mm, matrix=256×256, flip angle=60°.
A contouring program, Fujin Plus 08 version 3 (Tokyo, Japan), was used to trace the endocardial border of the LV myocardium for each slice of the heart over the entire cardiac cycle in order to determine ejection fraction (EF). This technique has previously been used to evaluate LV function after injection of embryonic stem cells to treat acute myocardial infarction . Following imaging, animals were euthanized and hearts harvested and processed for histology (including hematoxylin and eosin) and trichrome staining.
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