Effects of low-intensity pulsed focal ultrasound-mediated delivery of endothelial progenitor-derived exosomes in tMCAo stroke

 

Will your competent? doctor ensure human testing gets done? Or has the board of directors incompetence let your stroke medical 'professionals' DO NOTHING that will further stroke recovery?

Do you prefer your doctor and hospital incompetence NOT KNOWING? OR NOT DOING?

Effects of low-intensity pulsed focal ultrasound-mediated delivery of endothelial progenitor-derived exosomes in tMCAo stroke

Ahmet Alptekin¹Mohammad B. Khan²Mahrima Parvin¹Hasanul Chowdhury¹Sawaiz Kashif¹Fowzia A. Selina¹Anika Bushra¹Justin Kelleher¹Santu Ghosh³Dylan Williams²Emily Blumling²Roxan Ara⁴Asamoah Bosomtwi⁴Joseph A. Frank⁵Krishnan M. Dhandapani⁶Ali S. Arbab^1*†

• ¹Tumor Angiogenesis Laboratory, GCC, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, United States
• ²Department of Neurology, Medical College of Georgia, Augusta University, Augusta, GA, United States
• ³Department of Biostatistics, Medical College of Georgia, Augusta University, Augusta, GA, United States
• ⁴Small Animal Imaging Core, GCC, Medical College of Georgia, Augusta University, Augusta, GA, United States
• ⁵Laboratory of Diagnostic Radiology Research, Clinical Center, National Institutes of Health, Bethesda, MD, United States
• ⁶Department of Neurosurgery, Medical College of Georgia, Augusta University, Augusta, GA, United States

Introduction: Exosomes from different sources have been used for therapeutic purposes to target stroke and other disorders. However, exosomes from endothelial progenitor cells (EPCs) have not been tested in any stroke model, and in vivo bio-distribution study is lacking. Targeted delivery of IV-administered exosomes has been a significant challenge. Delivery of exosomes to the brain is a daunting task, and a blood–brain barrier (BBB)-penetrable peptide is being considered. However, the next step in practical treatment will be delivering naïve (unmodified) exosomes to the stroke site without destroying host tissues or disrupting BBB, or the membranes of the delivery vehicles. Low-intensity-pulsed focused ultrasound (LIPFUS) is approved for clinical use in the musculoskeletal, transcranial brain, and physiotherapy clinics. The objectives of the proposed studies were to determine whether LIPFUS-mediated increased delivery of EPC-derived exosomes enhances stroke recovery and functional improvement in mice with transient middle cerebral artery occlusion (tMCAo) stroke.

Methods: To enhance exosome delivery to the stroke area, we utilized LIPFUS. We evaluated stroke volume using MRI at different time points and conducted behavioral studies parallel to MRI to determine recovery. Ultimately, we studied brain tissue using immunohistochemistry to assess the extent of stroke and tissue regeneration.

Results and Discussion: In vivo, imaging showed a higher accumulation of EPC exosomes following LIPFUS without any damage to the underlying brain tissues, increased leakage of albumin, or accumulation of CD45+ cells. Groups of mice (14–16 months old) were treated with Vehicle (PBS), LIPFUS only, EPC-exosomes only, and LIPFUS+EPC-exosomes. LIPFUS + EPC exosomes groups showed a significantly decreased stroke volume on day 7, decreased FluoroJade+ cells, and significantly higher numbers of neovascularization in and around the stroke areas compared to that of other groups.

Introduction

Stroke is a significant cause of adult mortality and remains a leading contributor to adult disability. The majority of strokes are often ischemic (87%) resulting from a significant vascular occlusion due to a thromboembolic (TE) clot; therefore, the desired amount of stroke-salvaging drugs cannot be delivered to the core of the stroke (1, 2). In addition, the blood–brain barrier (BBB) is largely impermeable to most therapeutics. To date, IV tissue plasminogen activator (tPA) and/or endovascular thrombectomy (ET) are the only two Food and Drug Administration (FDA)-approved therapies to treat ischemic stroke. However, the recanalization of major vessels with IV-tPA/ET does not ensure adequate microvascular perfusion and recovery of tissue damage due to the associated risk of the “no-reflow” phenomenon that is exacerbated under stroke-related comorbidities such as aging, diabetes, or hypertension(So you haven't even attempted to solve Capillaries that don't open due to pericytes?)

. Preclinical modeling using the transient middle cerebral artery occlusion (tMCAo) model, validated in both mice and rats, can replicate confirmed recanalization and is widely accepted in stroke research (3). Thrombectomy in patients, and tMCAo in animals, assures that the test drug or treatment reaches the brain after ischemia and reperfusion (4). Thus, effective therapy and a new way of delivering therapeutic agents that can be safe in a larger comorbid stroke population and remain usable in multiple settings with or without IV-tPA/ET even after the therapeutic window are greatly needed.

Stem cell (or progenitor cell) treatments have shown to be successful in various preclinical models for different disease treatments, including stroke. Our previous studies showed the effectiveness of endothelial progenitor cells (EPCs) and neural stem cells (NSCs) in stroke models, with both in vivo magnetic resonance imaging (MRI) and functional behavioral studies showing improvement in stroke recovery (5–9). We postulate that both types of cells exerted a paracrine effect through exosomes, which might carry materials from their parental cells (10–12). Exosomes can interact with cells by fusion with the plasma membrane and subsequent endocytosis and release of their cargo, consisting of proteins, soluble factors, lipids, DNAs, microRNAs (miRNAs), and RNAs (13–16). Leveraging exosomes to harness the therapeutic potential of stem and progenitor cells would overcome the challenge that cell-based treatments face in clinical settings.

Exosomes are 30–150 nm in size and contain certain tetraspanins, heat shock proteins, biogenesis-related proteins, membrane transport and fusion proteins, nucleic acids, and lipids (17–19). Due to their biocompatibility, low toxicity, immunogenicity, permeability (including through the BBB), stability in biological fluids, and ability to accumulate in the lesions with higher specificity (20–26), investigators have used exosomes in different disease conditions in the brain, such as stroke, traumatic brain injury, and tumors (22, 27–30). Investigators have used exosomes and miRNA-rich exosomes collected from mesenchymal stem cells (MSCs) to enhance stroke recovery and showed that miRNA promoted neural plasticity and functional recovery (31, 32). Our group recently reported NSC-derived exosomes’ effect on improving tissue and functional recovery in the murine TE stroke model (33). However, exosomes from EPCs, which play an important role in vascular regeneration, have not been tested in any stroke model, and an in vivo bio-distribution study is lacking. Our published studies showed a dramatic reduction in stroke injury when EPCs were administered 24 h following stroke (5). Based on these results, we anticipate that EPCs-derived exosomes will cease the progression of stroke lesions and improve post-stroke outcomes if delivered efficiently to the site of stroke injury.

Targeted delivery of IV-administered exosomes has been a great challenge. Modification of the exosome surface to carry different ligands or peptides has been tried to increase delivery to target tissues (34, 35); however, the overall results are not encouraging (36, 37). Delivery of exosomes to the brain is a daunting task, and a BBB-penetrable peptide is being considered (38, 39). However, a method that can deliver naïve (unmodified) exosomes to the site of stroke without destroying host tissues, disrupting BBB, or affecting the membranes of the delivery vehicles (such as exosomes) would be a significant breakthrough.

High-Intensity Pulsed Focused ultrasound (HIPFUS) is being investigated to enhance permeability and retention (EPR) of nanoparticles/gene/plasmid/vectors to the sites of interest (40–45) but has been shown to cause widespread damage to local tissues including the brain (46). The basic mechanism behind HIPFUS’s effect is primarily through mechanical forces (acoustic radiation and acoustic cavitation) that increase the permeability of the vasculature, resulting in leakage of circulating nanoparticles, plasmids, or vectors into targeted sites and EPR (47–49). Low-intensity-pulsed ultrasound is approved for clinical use in musculoskeletal, central nervous system, and physiotherapy clinics. We have previously demonstrated that low-intensity-pulsed focused ultrasound (LIPFUS) increases the delivery of intravenously administered exosomes to stroke areas without causing damage to the brain (50). The objectives of the proposed studies are to determine whether LIPFUS-mediated increased delivery of EPC exosomes enhances stroke recovery and functional improvement in mice with tMCAo stroke.

In this study, we utilized EPC-derived exosomes for stroke treatment. To enhance exosome delivery to the stroke area, we employed LIPFUS without an ultrasound contrast agent microbubble/nanobubble infusion, which might temporarily disrupt the BBB, as described in our previous study (50). We assessed stroke volume using MRI at different time points and conducted behavioral studies parallel to MRI to evaluate recovery. In the end, we examined the brain tissue using immunohistochemistry studies to evaluate the extent of stroke and tissue regeneration. We found that EPC-derived exosome treatment reduced stroke volume 1 week after the stroke onset.