Terzic, André

Objectives: Peripheral arterial disease is a significant cause of morbidity and mortality. The goal of this study was to develop a novel treatment to promote angiogenesis. Platelets have been previously implicated in angiogenic events; however, platelet activation rather than presence of growth factors has been associated with new blood vessel formation. This study evaluated exosomes purified from activated platelets to determine their potential in promoting angiogenesis both in vitro and in vivo. Methods & Results: Under Current Good Manufacturing Practices, conditioned medium processed from apheresis platelet cultures yielded a purified exosome product (PEP) enriched for CD63, CD9 and Alix. In vitro, PEP drove HUVEC proliferation, migration, and vascular tube formation. Proteomic evaluation showed Flk1 (VEGFR2) was one of the key angiogenic proteins in PEP. HUVECs treated with Flk1 antibody neutralized PEP (anti-Flk1-PEP) showed reduced vascular tube formation compared to PEP. To evaluate in vivo angiogenesis, a rodent model of hindlimb ischemia was treated with sustained release PEP embedded in fibrin glue (PEP/TISSEEL) with blood flow evaluated by SPY angiography at 28 days post-treatment. Animals treated with PEP/TISSEEL showed improved blood flow with significant difference (p<0.001) as compared to sham, TISSEEL, and anti-Flk1-PEP/TISSEEL. Muscle tissue stained with α smooth muscle actin showed a significant increase (p<0.001) in vascular area in PEP/TISSEEL treated animals compared to TISSEEL alone. To further evaluate clinical translatability, a rabbit ear ischemia wound model was then treated with PEP/TISSEEL. Rabbit ears showed accelerated wound healing and restored blood flow on SPY angiography. Histologic evaluation of vascular area showed a significant increase in vascular area (p<0.05) compared to sham and TISSEEL alone animals. Conclusions: Exosome driven donation of Flk1 here promoted angiogenic events and was successfully translated into healing outcomes in the setting of two animal models of peripheral ischemia. This novel insight into biotherapy achievable pro-angiogenesis establishes a clinical-grade off-the-shelf exosome technology for the treatment of peripheral vascular disease.

AbstractA promising new strategy emerged in bone tissue engineering is to incorporate black phosphorus (BP) into polymer scaffolds, fabricating nanocomposite hydrogel platforms with biocompatibility, degradation controllability, and osteogenic capacity. BP quantum dot is a new concept and stands out recently among the BP family due to its tiny structure and a series of excellent characteristics. In this study, BP was processed into nanosheets of three different sizes via different exfoliation strategies and then incorporated into cross‐linkable oligo[poly(ethylene glycol) fumarate] (OPF) to produce nanocomposite hydrogels for bone regeneration. The three different BP nanosheets were designated as BP‐L, BP‐M, and BP‐S, with a corresponding diameter of 242.3 ± 90.0, 107.1 ± 47.9, and 18.8 ± 4.6 nm. The degradation kinetics and osteogenic capacity of MC3T3 pre‐osteoblasts in vitro were both dependent on the BP size. BP exhibited a controllable degradation rate, which increased with the decrease of the size of the nanosheets, coupled with the release of phosphate in vitro. The osteogenic capacity of the hydrogels was promoted with the addition of all BP nanosheets, compared with OPF hydrogel alone. The smallest BP quantum dots was shown to be optimal in enhancing MC3T3 cell behaviors, including spreading, distribution, proliferation, and differentiation on the OPF hydrogels. These results reinforced that the supplementation of BP quantum dots into OPF nanocomposite hydrogel scaffolds could potentially find application in the restoration of bone defects.

Evaluation of disease phenotypes and drug effects requires knowledge not only metabolite levels but also their turnover rates from which metabolic fluxes and status of the whole energetic system can be determined. Oxygen-18-assited 31 P NMR and mass spectrometric technique uniquely allow simultaneous measurements of phosphorus-containing metabolite levels and their turnover rates. This includes simultaneous recordings of 18 O-labeling rates reflecting ATP synthesis (γ-ATP) and ATP utilization (Pi), phosphotransfer fluxes through adenylate kinase (β-ATP/ADP), creatine kinase (CrP) and glycolytic pathways (G-6-P) as well as Krebs cycle associated mitochondrial nucleotide turnover (γ/β-GTP), substrate shuttle (G-3-P) and glycogen metabolism (G-1-P). Metabolomic and fluxomic profiling of hearts and body fluids of phosphotransfer enzyme deficient transgenic animals using 18 O-assisted GC/MS, 1 H and 18 O-assisted 31 P NMR indicate selective metabolic perturbations and adaptations in the whole energetic system. Adenylate kinase and creatine kinase deficiencies were associated with altered metabolomic profiles, increased mitochondrial capacities and redistribution of phosphotransfer flux through glycolytic and guanine nucleotide systems. These phosphometabolomic alterations were more expressed when animals were subjected to treadmill exercise, mitochondria-targeting drugs such as metformin and high-fat diet. Metabolite turnover rates are correlated with heart performance unveiling robustness and dynamics rearrangements in cell bioenergetics infrastructure. Glycerophosphate, G-3-P, metabolic dynamics is altered in transgenic animal models indicating defects in substrate shuttle and supply of reducing equivalents to mitochondria. This is of importance since G-3-P metabolic abnormalities and metabolic arrest are linked to human diseases such as sudden death syndrome. Thus, our study demonstrates that phosphometabolomic and fluxomic profiling is a valuable tool for metabolic phenotyping of transgenic animal models of human diseases as well as for plasma biomarker identification and monitoring of treatment efficacy and drug toxicity.

Adenylate kinase 2 (AK2) isoform in mitochondrial intermembrane/intracristal space provides a conduit facilitating ATP export and delivery to cellular ATPases. AK2+/- transgenic mice is viable while homozygous AK2-/- deficiency results in midgestation embryonic lethality. AK2-/- mouse embryonic fibroblasts (MEFs) have severely disrupted mitochondrial cristae structure and display low growth and proliferation potential. Protein knockdown using siRNA indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Transgenic AK2+/- hearts display greater changes in AMP/ATP ratio and AMPK activation during ischemia compared to wild type suggesting deficient tuning of AMP signals. Metabolomic profile of AK2+/- hearts subjected to myocardial ischemia indicate lower ability to maintain adenine and guanine nucleotide pools. AK2+/- mice have abnormal metabolomic response to treadmill exercise compared to wild type. Metabolomic profiling revealed that most significant plasma metabolites critical in the discrimination between wild type and AK2+/- phenotypes were adenosine, tyrosine, tryptophan and hypoxanthine indicating lower energetic potential of AK2+/- mice. AK2+/- hearts have increased mitochondrial creatine kinase CKmit content which could be viewed as a compensatory mechanism. Thus, AK2 is the first phosphotransfer enzyme which deficiency causes embryonic lethality indicating critical significance of catalyzed nucleotide exchange and conductive transfer in the narrow and crowded mitochondrial intermembrane/intracristal space.

Aims Bone marrow-derived mesenchymal stem cells obtained from bone marrow aspirate concentrate (BMAC) with platelet-rich plasma (PRP), has been used as an adjuvant to hip decompression. Early results have shown promise for hip preservation in patients with osteonecrosis (ON) of the femoral head. The purpose of the current study is to examine the mid-term outcome of this treatment in patients with precollapse corticosteroid-induced ON of the femoral head. Methods In all, 22 patients (35 hips; 11 males and 11 females) with precollapse corticosteroid-induced ON of the femoral head underwent hip decompression combined with BMAC and PRP. Mean age and BMI were 43 years (SD 12) and 31 kg/m² (SD 6), respectively, at the time of surgery. Survivorship free from femoral head collapse and total hip arthroplasty (THA) and risk factors for progression were evaluated at minimum five-years of clinical follow-up with a mean follow-up of seven years (5 to 8). Results Survivorship free from femoral head collapse and THA for any reason was 84% and 67% at seven years postoperatively, respectively. Risk factors for conversion to THA included a high preoperative modified Kerboul angle (grade 3 or 4) based on preoperative MRI (hazard ratio (HR) 3.96; p = 0.047) and corticosteroid use at the time of decompression (HR 4.15; p = 0.039). The seven-year survivorship for patients with grade 1 or 2 Kerboul angles for conversion to THA for articular collapse, and THA for any reason, were 96% and 72%, respectively, versus THA for articular collapse and THA for any reason in patients with grade 3 or 4 Kerboul angles of 40% (p = 0.003) and 40% (p = 0.032). Conclusion At seven years, hip decompression augmented with BMAC and PRP provided a 67% survivorship free from THA in patients with corticosteroid-induced ON. Ideal candidates for this procedure are patients with low preoperative Kerboul angles and can stop corticosteroid treatment prior to decompression. Cite this article: Bone Jt Open 2021;2(11):926–931.

Heart failure pathobiology is permissive to reparative intent. Regenerative therapies exemplify an emerging disruptive innovation aimed at achieving structural and functional organ restitution. However, mixed outcomes, complexity in use, and unsustainable cost have curtailed broader adoption, mandating the development of novel cardio-regenerative approaches. Lineage guidance offers a standardized path to customize stem cell fitness for therapy. A case in point is the molecular induction of the cardiopoiesis program in adult stem cells to yield cardiopoietic cell derivatives designed for heart failure treatment. Tested in early and advanced clinical trials in patients with ischemic heart failure, clinical grade cardiopoietic cells were safe and revealed therapeutic improvement within a window of treatment intensity and pre-treatment disease severity. With the prospect of mass customization, cardiopoietic guidance has been streamlined from the demanding, recombinant protein cocktail-based to a protein-free, messenger RNA-based single gene protocol to engineer affordable cardiac repair competent cells. Clinical trial biobanked stem cells enabled a systems biology deconvolution of the cardiopoietic cell secretome linked to therapeutic benefit, exposing a paracrine mode of action. Collectively, this new knowledge informs next generation regenerative therapeutics manufactured as engineered cellular or secretome mimicking cell-free platforms. Launching biotherapeutics tailored for optimal outcome and offered at mass production cost would contribute to advancing equitable regenerative care that addresses population health needs.

In situ cell recruitment is a promising regenerative medicine strategy with the purpose of tissue regeneration without stem cell transplantation. This chemotaxis-based strategy is aimed at ensuring a restorative environment through the release of chemokines that promote site-specific migration of healing cell populations. Stromal cell-derived factor-1α (SDF-1α) is a critical chemokine that can regulate the migration of mesenchymal stem cells (MSCs). Accordingly, here, SDF-1α-loaded microporous oligo[poly(ethylene glycol) fumarate]/bis[2-(methacryloyloxy)ethyl] phosphate composites (SDF-1α/OPF/BP) were engineered and probed. SDF-1α/OPF/BP composites were loaded with escalating SDF-1α concentrations, namely, 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml, and were cocultured with MSC. Scratching assay, Transwell assay, and three-dimensional migration model were utilized to assess the migration response of MSCs. Immunofluorescence staining of Runx2 and osteopontin (OPN), ELISA assay of osteocalcin (OCN) and alkaline phosphatase (ALP), and Alizarin Red S staining were conducted to assess the osteogenesis of MSCs. All SDF-1α/OPF/BP composites engendered a release of SDF-1α (>80%) during the first four days. SDF-1α released from the composites significantly promoted migration and osteogenic differentiation of MSCs documented by upregulated expression of osteogenic-related proteins, ALP, Runx2, OCN, and OPN. SDF-1α at 100 ng/ml was optimal for enhanced migration and osteogenic proficiency. Thus, designed SDF-1α/OPF/BP composites were competent in promoting the homing and osteogenesis of MSCs and thus offer a promising bioactive scaffold candidate for on-demand bone tissue regeneration.

Metabolic drugs frequently target mitochondrial functions and AMP signaling. Metformin, one of the most prescribed anti-diabetic medications, exerts its glucose lowering effect through mild interference with mitochondrial energy metabolism triggering homeostatic adenylate kinase (AK)-catalyzed AMP and downstream AMPK signaling. However, metabolomic signature of drug-induced “therapeutic mitochondrial toxicity" and energetic imbalance-induced AMP signaling are poorly understood. This work was aimed to determine metabolomic representation of metformin-induced mitochondrial and AMP signaling stress by comparing wild type and AK1-deficient mice with compromised AMP-signaling. Metabolomic profiling of plasma and urine samples from wild type mice treated with metformin revealed distinct metabolic signature suggesting altered energy, Krebs cycle, lipid and ketone body metabolism, with higher levels of creatinine, malate, α-ketoglutarate, FFA, glycerol, glycerol-3-phosphate, 3-hydroxybutyrate and hypoxanthine. Contrary to expectations glucose lowering effect of metformin was greater in energetically and AMP-signaling compromised AK1-/- mice. Metformin action on AK1 deficient mice was characterized by increased levels of creatinine, lactate, alanine, FFA, phosphoserine, acetoacetate, 3-hydroxybutyrate, malate and α-ketoglutarate indicating broader alterations in energy, glycolytic, Krebs cycle, ketone body, lipid and amino acid metabolism. At baseline, livers from AK1-/- mice had higher AMP/ADP and pAMPKα/AMPKα ratios compared to wild type. There were no significant sustained changes in global nucleotide ratios after metformin treatment. Overall metabolomic profile of AK1-/- mice indicate larger disturbances in energetic and substrate metabolic networks after metformin treatment compared to wild type. Thus, this study revealed specific metabolomic signatures of metformin therapeutic action and mitochondrial toxicity indicating that genetic and metabolic background modulate individual response to metformin treatment.

Hypoplastic Left Heart Syndrome (HLHS) is a complex multifactorial disease for which no definitive genetic causes have been found. Current genetic filtering strategies render lists of genes with unknown relevance in terms of pathogenesis. A complementary filter based on biological evidence would create a new approach to prioritize relevant candidate genes and mutations. In our study, 5 members of a nuclear family including a child with HLHS were evaluated using echocardiography and their genetic information was obtained through whole genome sequencing (WGS). Data filtering including rarity, functional impact and mode of inheritance was implemented, resulting in identification of 34 genes with recessive or de novo variants potentially involved in the pathogenesis of HLHS. Additionally, iPSC were derived from proband and parents and subjected to RNA-sequencing at the undifferentiated state and following spontaneous differentiation. Comparative transcriptional analyses identified genes differentially expressed in proband samples at each stage. These gene sets were used as an additional filter for the previously generated WGS data. This strategy revealed that out of 34 mutated genes originally identified, 10 displayed transcriptional differences in undifferentiated iPSC from the HLHS-affected individual while 16 out of 34 mutated genes showed significantly different expression levels in differentiated cells from proband. Furthermore, expression dynamics were studied during guided cardiac differentiation for the 9 genes fulfilling all applied criteria. Two genes not previously linked to HLHS, ELF4 and HSPG2 were found to behave significantly different in HLHS-iPSC when compared to control counterparts. In summary, filtering WGS data according to a new layer of transcriptional information that leverages iPSC plasticity allows prioritization of genes associated with HLHS in an in vitro model of disease.