Mid-Systolic Notching of the Pulmonary Valve Doppler Signal is Highly Associated with Pulmonary Hypertension.

Mid-systolic notching (MSN) of the pulmonary valve Doppler signal represents a reflected systolic pressure wave from the pulmonary vasculature and is often seen in pulmonary hypertension (PH). We hypothesize that MSN is associated with a higher pulmonary vascular resistance (PVR) and mean pulmonary artery pressure (mPAP), and a diagnosis of PH in pediatric patients. This was a retrospective study of patients ≤ 18 years who had an echocardiogram obtained ≤ 30 days before catheterization for suspected PH. MSN was defined as an indentation in the initial two thirds of the systolic Doppler signal. PH was defined as mPAP > 20 mmHg and PVR ≥ 3.0 Wu  m2. Subgroups (MSN vs. normal) were compared. Receiver operator characteristic determined a continuous variable's discriminatory ability for a diagnosis of PH. Reproducibility of MSN was assessed. In total, 90 patients (73 with congenital heart disease) were included, of which 36 had MSN and 54 were normal. MSN patients were more likely to have PH, and had significantly higher mPAP, PVR, and lower pulmonary stroke volume. The presence of MSN had good discriminatory ability for PH diagnosis. The presence of MSN had high specificity (96%) for PH, whereas sensitivity was lower (54%). Reproducibility was 100% for MSN. MSN is a simple, highly reproducible echocardiographic metric associated with higher mPAP and PVR. When present, there is a high likelihood a diagnosis of PH confirmed by catheterization. Incorporation of MSN into imaging protocols may be useful. MSN appears worthy of further investigation in pediatric patients with suspected PH.

Senning With Aortic Translocation and Anatomic Repair for Congenitally Corrected Transposition.

BACKGROUND: Anatomic repair for congenitally corrected transposition of the great arteries with ventricular septal defect (VSD) and pulmonic stenosis has been accomplished with atrial switch and Rastelli. Aortic translocation offers a direct left ventricular outflow without an extraanatomic right ventricular-to-pulmonary conduit, which may lead to decreased reoperations. We reviewed our entire experience performing Senning with aortic translocation (SAT). METHODS: From 2007 to 2017, 8 patients (mean age, 14.1 months; size, 8.86 kg) underwent SAT. Associated anomalies included situs inversus (n = 2), dextrocardia (n = 6), multiple muscular VSDs (n = 2), abnormal or straddling atrioventricular valve chords (n = 5), and branch pulmonary artery stenosis (n = 3). Four of 8 had previous systemic arterial shunts. Mean cardiopulmonary bypass was 487 minutes, and mean cardiac ischemic time was 307 minutes. Additional procedures included repair of branch pulmonary artery stenoses and closure of multiple muscular VSDs. RESULTS: There was no hospital death. One patient was supported with extracorporeal membrane oxygenation because of junctional tachycardia on postoperative day 5. One patient required pacemaker placement for first-degree heart block. Median hospital length of stay was 31 days. Mean length of follow-up was 52 months. All patients remain well with mild or no aortic regurgitation. The first patient underwent a repeat surgical operation for pulmonary venous baffle obstruction 2 years after SAT. CONCLUSIONS: Despite the technical complexity, patient outcomes have been satisfactory. We believe SAT provides a superior anatomic repair in these complex defects. Longer-term follow-up is needed regarding late intervention.

Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization.

The use of nanomaterials as carriers for the delivery of growth factors has been applied to a multitude of applications in tissue engineering. However, issues of toxicity, stability, and systemic effects of these platforms have yet to be fully understood, especially for cardiovascular applications. Here, we proposed a delivery system composed of poly(dl-lactide- co-glycolide) acid (PLGA) and porous silica nanoparticles (pSi) to deliver vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The tight spatiotemporal release of these two proteins has been proven to promote neovascularization. In order to minimize tissue toxicity, localize the release, and maintain a stable platform, we conjugated two formulations of PLGA-pSi to electrospun (ES) gelatin to create a combined ES patch releasing both PDGF and VEGF. When compared to freely dispersed particles, the ES patch cultured in vitro with neonatal cardiac cells had significantly less particle internalization (2.0 ± 1.3%) compared to free PLGA-pSi (21.5 ± 6.1) or pSi (28.7 ± 2.5) groups. Internalization was positively correlated to late-stage apoptosis with PLGA-pSi and pSi groups having increased apoptosis compared to the untreated group. When implanted subcutaneously, the ES patch was shown to have greater neovascularization than controls evidenced by increased expression of α-SMA and CD31 after 21 days. Quantitative reverse transcription-polymerase chain reaction results support increased angiogenesis by the upregulation of VEGFA, VEGFR2, vWF, and COL3A1, exhibiting a synergistic effect with the release of VEGF-A164 and PDGF-BB after 21 days in vivo. The results of this study proved that the ES patch reduced cellular toxicity and may be tailored to have a dual release of growth factors promoting localized neovascularization.

Controlled Release of Small Molecules for Cardiac Differentiation of Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have been shown to differentiate to functional cardiomyocytes (CM) with high efficiency through temporally controlled inhibition of the GSK3/Wnt signaling pathways. In this study, we investigated the ability of temporally controlled release of GSK3/Wnt small-molecule inhibitors to drive cardiac differentiation of iPSC without manual intervention. Porous silica particles were loaded with GSK3 inhibitor CHIR99021 or Wnt inhibitor IWP2, and the particles containing IWP2 were coated with 5 wt% poly(lactic-co-glycolic acid) 50:50 to delay release by ∼72 h. iPSCs reprogrammed through mRNA transfection were cultured with these particles up to 30 days. High-performance liquid chromatography suggests a burst release of CHIR99021 within the first 24 h and a delayed release of IWP2 after 72 h. Annexin V/propidium iodide staining did not show a significant effect on apoptosis or necrosis rates. Cultured cells upregulated both early (Nkx 2.5, Isl-1) and late (cTnT, MHC, Cx43) cardiac markers, assayed with a quantitative real-time polymerase chain reaction, and began spontaneous contraction at 3.0 ± 0.6 Hz at 15-21 days after the start of differentiation. CM had clear sarcomeric striations when stained for ß-myosin heavy chain, and showed expression and punctate membrane localization of gap junction protein Connexin43. Calcium and voltage-sensitive imaging showed both action potential and calcium transients typical of immature CM. This study showed that the cardiac differentiation of pluripotent stem cells can be directed by porous silica vectors with temporally controlled release of small-molecule inhibitors. These results suggest methods for automating and eliminating variability in manual maintenance of inhibitor concentrations in the differentiation of pluripotent stem cells to CM.

Biomimetic Tissue Engineering: Tuning the Immune and Inflammatory Response to Implantable Biomaterials.

Regenerative medicine technologies rely heavily on the use of well-designed biomaterials for therapeutic applications. The success of implantable biomaterials hinges upon the ability of the chosen biomaterial to negotiate with the biological barriers in vivo. The most significant of these barriers is the immune system, which is composed of a highly coordinated organization of cells that induce an inflammatory response to the implanted biomaterial. Biomimetic platforms have emerged as novel strategies that aim to use the principle of biomimicry as a means of immunomodulation. This principle has manifested itself in the form of biomimetic scaffolds that imitate the composition and structure of biological cells and tissues. Recent work in this area has demonstrated the promising potential these technologies hold in overcoming the barrier of the immune system and, thereby, improve their overall therapeutic efficacy. In this review, a broad overview of the use of these strategies across several diseases and future avenues of research utilizing these platforms is provided.

Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells.

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and ß-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and ß-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.

Full-Thickness Heart Repair with an Engineered Multilayered Myocardial Patch in Rat Model.

In a rat model of right free wall replacement, the transplantation of an engineered multilayered myocardial patch fabricated from a polycaprolactone membrane supporting a chitosan/heart matrix hydrogel induces significant muscular and vascular remodeling and results in a significantly higher right ventricular ejection fraction compared to use of a commercially available pericardium patch.

Platelet-derived growth-factor-releasing aligned collagen-nanoparticle fibers promote the proliferation and tenogenic differentiation of adipose-derived stem cells.

In order to enhance the healing potential of an injured tendon, we have prepared a novel biomimetic aligned collagen-nanoparticle (NP) composite fiber using an electrochemical process. The aligned collagen-NP composite fiber is designed to affect the cellular activity of adipose-derived stem cells (ADSCs) through two different ways: (i) topographic cues from the alignment of collagen fibril and (ii) controlled release of platelet-derived growth factors (PDGFs) from the NPs. PDGF released from collagen-NP fibers significantly enhanced the proliferation of ADSCs when tested for up to 7 days. Moreover, compared to random collagen fibers with PDGFs, aligned collagen-NP fibers significantly promoted the desirable tenogenic differentiation of ADSCs, as evidenced by an increased level of tendon markers such as tenomodulin and scleraxis. On the other hand, no undesirable osteogenic differentiation, as measured by the unchanged level of alkaline phosphatase and osteocalcin, was observed. Together, these results indicate that the aligned collagen-NP composite fiber induced the tenogenic differentiation of ADSCs through both a topographic cue (aligned collagen fibril) and a chemical cue (PDGF released from NPs). Thus, our novel aligned collagen-NP composite fiber has a significant potential to be used for tendon tissue engineering and regeneration.

Wnt/ß-catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts.

The BMP and Wnt/ß-catenin signaling pathways cooperatively regulate osteoblast differentiation and bone formation. Although BMP signaling regulates gene expression of the Wnt pathway, much less is known about whether Wnt signaling modulates BMP expression in osteoblasts. Given the presence of putative Tcf/Lef response elements that bind ß-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/ß-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/ß-catenin signaling is active in various osteoblast or osteoblast precursor cell lines, including MC3T3-E1, 2T3, C2C12, and C3H10T1/2 cells. Furthermore, crosstalk between the BMP and Wnt pathways affected BMP signaling activity, osteoblast differentiation, and bone formation, suggesting Wnt signaling is an upstream regulator of BMP signaling. Activation of Wnt signaling by Wnt3a or overexpression of ß-catenin/TCF4 both stimulated BMP2 transcription at promoter and mRNA levels. In contrast, transcription of BMP2 in osteogenic cells was decreased by either blocking the Wnt pathway with DKK1 and sFRP4, or inhibiting ß-catenin/TCF4 activity with FWD1/ß-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/ß-catenin transactivation of BMP2 transcription is directly mediated through the Tcf/Lef response elements in the BMP2 promoter. These results, which demonstrate that the Wnt/ß-catenin signaling pathway is an upstream activator of BMP2 expression in osteoblasts, provide novel insights into the nature of functional cross talk integrating the BMP and Wnt/ß-catenin pathways in osteoblastic differentiation and maintenance of skeletal homeostasis.