Regenerative Growth Research Articles

Long-term fate tracking and quantitative analyzing of nanoparticles in stem cells with bright-field microscopy

The evolution of nanotechnology gives impetus to the regenerative medicine growth, which has led to the great research interest in studying the interaction between stem cells and nanoparticles (NPs). The study of intracellular fate of NPs is generally consisted of two aspects: the dynamic tracking of NPs (NPs localization and transportation) and the quantification of intracellular NPs content. However, there is a lack of tools that could satisfy both requirements at the same time. In this work, Prussian blue nanoparticles (PBNPs) were chosen as model probes to study the fate of NPs in living cells. By combining the live cell imaging system with proposed quantification method (pixel-based colorimetry method), we realized a long-term monitoring and dynamic quantitation of PBNPs in living stem cells at the single-cell level. NPs fate tracking and quantitative analysis have visualized the asymmetrical inheritance of PBNPs in two daughter cells during mitosis and the reduction of PBNPs during interphase, which is mainly attributed by lysosomal digestion. As demonstrated by our results, we identified three factors that contributed to the PBNPs load change within one cell cycle: asymmetric cell inheritance (92.78%; 59.19% vs 33.59% in two cells), lysosomal digestion (6.93%), and exocytosis (0.29%). In summary, this study provided detailed insight of NPs’ fate in cell cycles which is essential for rational design of NPs in the future.

Obesity in Angiogenesis and Lung Regeneration

Obesity is associated with impairment of wound healing and tissue regeneration. Angiogenesis, the formation of new blood capillaries, plays a key role in organ regeneration and repair. We and other groups have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that obesity is accompanied by endothelial cell dysfunction. The aim of this project is to investigate the effects of obesity on post‐PNX lung vascular and alveolar morphogenesis. We have found that post‐PNX regenerative lung growth is inhibited in Lepob/ob obese mice compared to Lepob/+ mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the lung tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX Lepob/ob mice. The levels of adiponectin, one of the adipokines that exhibits pro‐angiogenic and vascular protective properties, increase in the remaining mouse lungs after unilateral PNX, while these effects are attenuated in Lepob/ob obese mice. Regenerative lung growth, vascular and alveolar morphogenesis, and VEGF levels in the post‐PNX mouse lungs are inhibited in adiponectin knockout mice. These results suggest that obesity inhibits post‐PNX regenerative lung growth through adiponectin‐VEGF signaling. Modulation of adiponectin‐VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese people.

Hydrostatic Pressure Controls Angiogenesis through Endothelial YAP1 during Lung Regeneration

Pulmonary artery (PA) pressure increases during lung growth after unilateral pneumonectomy (PNX). The objective of this study is to understand the mechanism by which increases in PA pressure control angiogenesis during lung regeneration. Mechanosensitive transcriptional co‐activator, yes‐associated protein (YAP1), in endothelial cells (ECs) is necessary for angiogenesis during post‐PNX lung growth. We investigate whether increases in PA pressure following PNX controls angiogenesis through YAP1. When hydrostatic pressure is applied to human pulmonary arterial ECs (HPAECs), the expression of YAP1, transcription factor TEAD1, and angiogenic factor receptor Tie2 increases, while these effects are inhibited when HPAECs are treated with YAP1 siRNA or YAP1S94A mutant that fails to bind to TEAD1. Hydrostatic pressure also stimulates DNA synthesis and cell migration in HPAECs, while YAP1 knockdown or YAP1S94A mutant inhibits the effects. Gene enrichment analysis reveals that the levels of extracellular matrix or cell adhesion molecules are altered in post‐PNX mouse lung ECs, which interact with YAP1. These results suggest that increases in PA pressure stimulate angiogenesis through YAP1 during regenerative lung growth.

Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14–22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Characterization of a Novel Aspect of Tissue Scarring Following Experimental Spinal Cord Injury and the Implantation of Bioengineered Type-I Collagen Scaffolds in the Adult Rat: Involvement of Perineurial-like Cells?

Numerous intervention strategies have been developed to promote functional tissue repair following experimental spinal cord injury (SCI), including the bridging of lesion-induced cystic cavities with bioengineered scaffolds. Integration between such implanted scaffolds and the lesioned host spinal cord is critical for supporting regenerative growth, but only moderate-to-low degrees of success have been reported. Light and electron microscopy were employed to better characterise the fibroadhesive scarring process taking place after implantation of a longitudinally microstructured type-I collagen scaffold into unilateral mid-cervical resection injuries of the adult rat spinal cord. At long survival times (10 weeks post-surgery), sheets of tightly packed cells (of uniform morphology) could be seen lining the inner surface of the repaired dura mater of lesion-only control animals, as well as forming a barrier along the implant–host interface of the scaffold-implanted animals. The highly uniform ultrastructural features of these scarring cells and their anatomical continuity with the local, reactive spinal nerve roots strongly suggest their identity to be perineurial-like cells. This novel aspect of the cellular composition of reactive spinal cord tissue highlights the increasingly complex nature of fibroadhesive scarring involved in traumatic injury, and particularly in response to the implantation of bioengineered collagen scaffolds.

Mesenchymal stem cells protect against acetaminophen hepatotoxicity by secreting regenerative cytokine hepatocyte growth factor

BackgroundAcetaminophen (APAP) overdose is a major cause of the morbidity of acute liver failure. The current clinically approved treatment for APAP poisoning, N-acetylcysteine (NAC), has a limited therapeutic window, and prolonged treatment with NAC delays liver regeneration. Mesenchymal stem cells (MSCs) also have therapeutic effects on APAP-induced mouse liver failure, but whether the effects are comparable to those of NAC has not been determined, and the mechanism still needs further exploration.MethodsFasted C57BL/6 mice that received 500 mg/kg APAP were treated intravenously with 300 mg/kg NAC or different amounts of MSCs at 2 h after APAP to investigate survival, hepatocyte necrosis and neutrophil/macrophage recruitment. In vitro co-culture was performed to study the anti-necrotic effects of MSCs on the APAP-injured hepatocyte cell line L-O2.ResultsMSCs dose-dependently rescued the C57BL/6J mice from APAP-induced liver failure, with 87.5% of MSCs (1 × 106) surviving similar to that of NAC (90%). MSC has similar effects on reduced hepatocyte necrosis and granulocytic myeloid-derived suppressor cells (MDSC) infiltration but enhanced the proportion of regenerative monocytic MDSC when compared to NAC. Mechanistically, MSCs attenuate hepatocyte necrosis by secreting hepatocyte growth factor (HGF). When HGF was knocked down, the protective effects of MSCs were reduced on APAP-induced hepatocyte necrosis and mouse liver failure.ConclusionsMSCs are comparable to NAC against APAP-induced liver failure by secreting HGF with less regenerative retardation concerns, thus facilitating the application of MSCs in clinical therapy for APAP liver failure.

Circular economy as a new model of Ukrainian economy development in the european integration process

Methods. The methodological basis of the study was the concept of sustainable development, which considers socio-economic development as social change that occurs within the limits determined by the ability of ecosystems to recover, absorb pollution and support the lives of present and future generations. Specific research methods used are as follows: the method of abstraction – in establishing the essence of the circular model of the economy; functional analysis – in highlighting the relationship of European integration processes and the transition to a circular economy of Ukraine; comparative studies – while highlighting the benefits of the transition of the Ukrainian economy to a model of circular economy. Results. The central theme of the study was a significant increase in resource efficiency in the EU, which involves the transition to a circular economy and means ease of recycling, reuse, dismantling and recovery and should replace the traditional linear model that has dominated the economy so far. It is investigated that in order to save resources in the EU it is necessary to move to the model of regenerative growth. This means not using more than the planet provides in real time, reducing resource consumption and doubling the reuse of materials in the next decade. In this regard, the European Green Course (EGC) was analyzed in terms of opportunities and threats to Ukraine in key areas: energy, industry, transport, agricultural policy, biodiversity, pollution, finance, trade. Recommendations on using opportunities and avoiding threats to major stakeholders in Ukraine and the EU are provided. Novelty. The new EU policy and the policy of Ukraine in the field of circular economy are analyzed. Taking into account the best European practices, recommendations were given to Ukraine on the transition from the traditional (linear) economy to the new model. It is established that in general Ukraine's policy in the field of circular economy should be formed and implemented comprehensively in the context of European trends. Practical value. In the study is the introduction of the principles of the circular economy in Ukraine with the simultaneous introduction of EGC, which together with the modernization of production should have a positive economic effect - increase the competitiveness of domestic production and promote economic development.

Hydrostatic Pressure Controls Angiogenesis Through Endothelial YAP1 During Lung Regeneration.

Pulmonary artery (PA) pressure increases during lung growth after unilateral pneumonectomy (PNX). Mechanosensitive transcriptional co-activator, yes-associated protein (YAP1), in endothelial cells (ECs) is necessary for angiogenesis during post-PNX lung growth. We investigate whether increases in PA pressure following PNX control-angiogenesis through YAP1. When hydrostatic pressure is applied to human pulmonary arterial ECs (HPAECs), the expression of YAP1, transcription factor TEAD1, and angiogenic factor receptor Tie2 increases, while these effects are inhibited when HPAECs are treated with YAP1 siRNA or YAP1S94A mutant that fails to bind to TEAD1. Hydrostatic pressure also stimulates DNA synthesis, cell migration, and EC sprouting in HPAECs, while YAP1 knockdown or YAP1S94A mutant inhibits the effects. Gene enrichment analysis reveals that the levels of genes involved in extracellular matrix (ECM), cell adhesion, regeneration, or angiogenesis are altered in post-PNX mouse lung ECs, which interact with YAP1. Exosomes are known to promote tissue regeneration. Proteomics analysis reveals that exosomes isolated from conditioned media of post-PNX mouse lung ECs contain the higher levels of ECM and cell-adhesion proteins compared to those from sham-operated mouse lung ECs. Recruitment of host lung ECs and blood vessel formation are stimulated in the fibrin gel containing exosomes isolated from post-PNX mouse lung ECs or pressurized ECs, while YAP1 knockdown inhibits the effects. These results suggest that increases in PA pressure stimulate angiogenesis through YAP1 during regenerative lung growth.

Promoting axon regeneration in the central nervous system by increasing PI3-kinase signaling.

Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system. Axons in the central nervous system fail to regenerate, meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences. In 2008, genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve. PTEN is a phosphatase that opposes the actions of PI3-kinase, a family of enzymes that function to generate the membrane phospholipid PIP3 from PIP2 (phosphatidylinositol (3,4,5)-trisphosphate from phosphatidylinositol (4,5)-bisphosphate). Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase, and was initially demonstrated to promote axon regeneration by signaling through mTOR. More recently, additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability. This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3, and considers them in relation to both developmental and regenerative axon growth. We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability, and describe how these are affected by signaling through PI3-kinase. We highlight the recent finding of a developmental decline in the generation of PIP3 as a key reason for regenerative failure, and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system. Finally, we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.

Inventory and Comparison of Performance Indicators in Circular Economy Roadmaps of the European Countries

European Union (EU) is in the process of transformation toward a circular economy (CE), which is a regenerative growth model that gives back to the planet more than it takes. Member States are key players in this process; therefore, next to the EU Action Plan, they developed national strategies (roadmaps) toward the CE. The paper presents an inventory of performance indicators, which have been indicated in the selected CE national strategies. The EU countries strongly underline the necessity to access the transformation progress with the use of CE indicators at different levels (macro, meso, or/and micro) or for different actions or objectives. There is a lack of one universal indicator which measures a level of the CE transformation at the national level due to the complexity of this issue and the diversity of key sectors and economic actors in the individual countries. There are some proposed indicators that could be used in other countries, to shape national monitoring frameworks by policy-makers. However, to access the transformation process on the European level showing the comparison between different countries, it is recommended to use the CE monitoring framework because it contains the set of CE indicators that provide uniform data and holistic view of all countries in Europe.

Porous Nanomaterials Targeting Autophagy in Bone Regeneration.

Porous nanomaterials (PNMs) are nanosized materials with specially designed porous structures that have been widely used in the bone tissue engineering field due to the fact of their excellent physical and chemical properties such as high porosity, high specific surface area, and ideal biodegradability. Currently, PNMs are mainly used in the following four aspects: (1) as an excellent cargo to deliver bone regenerative growth factors/drugs; (2) as a fluorescent material to trace cell differentiation and bone formation; (3) as a raw material to synthesize or modify tissue engineering scaffolds; (4) as a bio-active substance to regulate cell behavior. Recent advances in the interaction between nanomaterials and cells have revealed that autophagy, a cellular survival mechanism that regulates intracellular activity by degrading/recycling intracellular metabolites, providing energy/nutrients, clearing protein aggregates, destroying organelles, and destroying intracellular pathogens, is associated with the phagocytosis and clearance of nanomaterials as well as material-induced cell differentiation and stress. Autophagy regulates bone remodeling balance via directly participating in the differentiation of osteoclasts and osteoblasts. Moreover, autophagy can regulate bone regeneration by modulating immune cell response, thereby modulating the osteogenic microenvironment. Therefore, autophagy may serve as an effective target for nanomaterials to facilitate the bone regeneration process. Increasingly, studies have shown that PNMs can modulate autophagy to regulate bone regeneration in recent years. This paper summarizes the current advances on the main application of PNMs in bone regeneration, the critical role of autophagy in bone regeneration, and the mechanism of PNMs regulating bone regeneration by targeting autophagy.

Lipid-Based Drug Delivery Systems in Regenerative Medicine.

The most important goal of regenerative medicine is to repair, restore, and regenerate tissues and organs that have been damaged as a result of an injury, congenital defect or disease, as well as reversing the aging process of the body by utilizing its natural healing potential. Regenerative medicine utilizes products of cell therapy, as well as biomedical or tissue engineering, and is a huge field for development. In regenerative medicine, stem cells and growth factor are mainly used; thus, innovative drug delivery technologies are being studied for improved delivery. Drug delivery systems offer the protection of therapeutic proteins and peptides against proteolytic degradation where controlled delivery is achievable. Similarly, the delivery systems in combination with stem cells offer improvement of cell survival, differentiation, and engraftment. The present review summarizes the significance of biomaterials in tissue engineering and the importance of colloidal drug delivery systems in providing cells with a local environment that enables them to proliferate and differentiate efficiently, resulting in successful tissue regeneration.

Semaphorin 3A-Glycosaminoglycans Interaction as Therapeutic Target for Axonal Regeneration.

Semaphorin 3A (Sema3A) is a cell-secreted protein that participates in the axonal guidance pathways. Sema3A acts as a canonical repulsive axon guidance molecule, inhibiting CNS regenerative axonal growth and propagation. Therefore, interfering with Sema3A signaling is proposed as a therapeutic target for achieving functional recovery after CNS injuries. It has been shown that Sema3A adheres to the proteoglycan component of the extracellular matrix (ECM) and selectively binds to heparin and chondroitin sulfate-E (CS-E) glycosaminoglycans (GAGs). We hypothesize that the biologically relevant interaction between Sema3A and GAGs takes place at Sema3A C-terminal polybasic region (SCT). The aims of this study were to characterize the interaction of the whole Sema3A C-terminal polybasic region (Sema3A 725–771) with GAGs and to investigate the disruption of this interaction by small molecules. Recombinant Sema3A basic domain was produced and we used a combination of biophysical techniques (NMR, SPR, and heparin affinity chromatography) to gain insight into the interaction of the Sema3A C-terminal domain with GAGs. The results demonstrate that SCT is an intrinsically disordered region, which confirms that SCT binds to GAGs and helps to identify the specific residues involved in the interaction. NMR studies, supported by molecular dynamics simulations, show that a new peptoid molecule (CSIC02) may disrupt the interaction between SCT and heparin. Our structural study paves the way toward the design of new molecules targeting these protein–GAG interactions with potential therapeutic applications.

Toward Low-Carbon European Union Society: Young Poles’ Perception of Climate Neutrality

(1) Introduction: The European Union is a global leader in the transition to a low-carbon economy. The community’s population has already reduced greenhouse gas emissions by 22%, while the gross domestic product (GDP) in its member states increased by 58% between 1990 and 2017. The Union has shown that economic growth is possible in the link between politics, nature, and the citizens’ eco-empathy. It has implications for both the European and global economy, infrastructure, food production, public health, and biodiversity, the formula for political stability. The hallmark of the European Union is the blending of politics, culture, and nature in its quest for climate neutrality. The community’s horizon is a zero-emission economy by 2050. (2) Theoretical framework: The cognitive assumptions of the article are the following theses: 1. The Union strives to accelerate the transition process to a regenerative growth model using technologies for obtaining and distributing energy for individual and collective needs; 2. The Union and the European citizens want to hand over more to planet Earth than they take away from it, thus making progress toward keeping resource consumption within planetary limits. 3. The Union aims to reduce its consumption footprint and double the rate of applying closed-loop materials in the next decade. Finally, it fits in with the organization’s policy projections. (3) Methodology and research results: Qualitative and quantitative research methods were used in the research process. The literature has been analyzed on the subject and the applicable legal acts, making it possible to classify, generalize, describe, and systematize the facts collected during the research. A survey was conducted on 1106 students, and in-depth interviews were conducted with three energy experts: Krzysztof Tomaszewski (the University of Warsaw, researcher and lecturer on energy security issues), Dariusz Pachniewski (inventor in energy sector, businessman in hydrogen energy sector), and Tomoho Umeda (President of Polish Chamber of Commerce). The research aimed to identify the environmental and energy awareness, knowledge of renewable energy sources, and opinions of young Poles on their use in two areas: home and work. (4) Discussion: The reinforced narrative of creating a low-carbon society, a green economy, was adapted and evaluated for innovative individual and collective approaches in the research conducted in this paper. In the quantitative and qualitative samples, the assumptions made were double-checked. The former verified the students’ views, while the latter, the experts’ views. The procedure established the directions of knowledge evolution and approach to technologies and innovations among students of technical faculties related to the energy sector. There were existing individual and collective mental constructs on energy transition and climate neutrality identified. (5) Conclusions: The surveys conducted among university students of energy-related majors and specialties and experts revealed important information. First, it concerned the way knowledge is communicated and how it is interpreted; second, informing and implementing the European Union’s climate policy; third, the creation of a low-carbon society; fourth, the perception of climate neutrality among young Poles, and finally, preferences in energy generation and use in homes and businesses.

A novel in vitro assay for peripheral nerve-related cell migration that preserves both extracellular matrix-derived molecular cues and nanofiber-derived topography

BackgroundMolecular composition and topography of the extracellular matrix (ECM) influence regenerative cell migration following peripheral nerve injury (PNI). Advanced tissue engineering strategies for the repair of neurotmesis-type PNI include the development of nanofiber-containing implantable scaffolds that mimic features of the ECM to orchestrate regenerative growth. Reliable and quantifiable in vitro assays are required to assess the ability of such substrates to influence migration of the cell types of interest. However, most popular migration assays monitor cell migration into a cell exclusion zone (CEZ) but have dubious abilities to preserve the molecular and topographical cues of the substrate. New methodElastic band spacers (EBS), a simple, economical and standardized technique for the generation of well-defined CEZ based on the use of commercially available elastic bands, are introduced. ResultsEBS could sufficiently preserve ECM-derived molecular and poly(ε-caprolactone) (PCL) nanofiber-derived topographical cues. The application of EBS in the absence and presence of nanofiber-derived topographical cues was validated using perineurial cells and Schwann cells, both known to play key roles in peripheral nerve regeneration. Comparison with existing methodsIn contrast to EBS, commercial silicone inserts and the popular scratch assay caused substantial ECM substrate disruption, thereby preventing these techniques from being included in further investigations employing deposition of PCL nanofibers and cell migration analysis. ConclusionsEBS represent a useful addition to the existing repertoire of migration assays offering significant benefits in terms of substrate preservation. The simplicity and economy of the approach make it immediately accessible to research groups at minimal extra expense.

Phenotypic Screening Following Transcriptomic Deconvolution to Identify Transcription Factors Mediating Axon Growth Induced by a Kinase Inhibitor.

After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors' combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.

Metabolite Profiling in Arabidopsisthaliana with Moderately Impaired Photorespiration Reveals Novel Metabolic Links and Compensatory Mechanisms of Photorespiration

Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsis thaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as 13C- and 14C-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism.

Obesity in Angiogenesis and Regenerative Lung Growth after Pneumonectomy

Obese population is rapidly growing worldwide. Obesity is associated with impairment of wound healing and tissue regeneration. Angiogenesis, the formation of new blood capillaries, plays a key role in organ regeneration and repair. We and other groups have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that obesity is accompanied by endothelial cell dysfunction. However, the effects of obesity on lung regeneration remain unclear. Compensatory lung growth and vascular and alveolar morphogenesis after PNX are suppressed in leptin-deficient ob/ob mice treated with high-fat diet compared to that in control lean mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the lung tissue collected from post-PNX mice compared to those from sham-operated control mice, while these effects are attenuated in post-PNX leptin-deficient ob/ob mice with high-fat diet. Among various VEGF isoforms, VEGF164, but not VEGF188 and VEGF120, increases in the post-PNX mouse lungs, while these effects are inhibited in obese mice. These results suggestthat obesity inhibits post-PNX regenerative lung growth through VEGF164 signaling. Modulation of VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese people.

Role of Lymphangiogenesis in Lung Regeneration

Lymphangiogenesis, the formation of lymphatic vessels, plays key roles in embryonic development and wound healing. We and other groups have reported that impaired blood vessel formation suppresses compensatory lung growth after pneumonectomy (PNX). However, the role of lymphatics in regenerative lung growth remains unknown. Here we find that lymphangiogenesis is stimulated in the mouse lung after PNX and expression of PROX1, which plays key roles in lymphatic development, and a major lymphatic growth factor, VEGFR3 is higher in the post-PNX mouse lungs compared to that in sham-operated control mice. Diphtheria toxin-induced depletion of lymphatic vessels or treatment with VEGFR3 inhibitor inhibits post-PNX lung growth. VEGFC stimulates lymphatic vessel formation in the gel implanted on the mouse lungs, while VEGFR3 inhibitor inhibits the effects. These results suggest that lymphangiogenesis is stimulated during lung growth after PNX through VEGFC-VEGFR3 signaling. Modulation of lymphangiogenesis may provide a novel strategy for lung regeneration.

ROBO/SLIT in Obesity‐dependent Changes in Angiogenesis and Lung Regeneration

More than 35 million people in the US suffer from chronic lung diseases, and a number of these patients have pre-existing conditions including obesity. Although lung transplantation is one of the options for end-stage lung diseases, due to the shortage of donors and low survival rate, this approach is suboptimal. In particular, there is an association between obesity and a poor outcome in lung transplantation. Thus, stimulating the regenerative ability of the lungs could be one of the therapeutic strategies for end-stage lung diseases in obese patients. Angiogenesis –the formation of new blood vessels- plays an important role in lung regeneration. Obesity is accompanied by endothelial cell dysfunction. However, the effects of obesity on lung vascular and alveolar regeneration remain unclear. Here we found that compensatory lung growth after unliteral pneumonectomy (PNX) was inhibited in leptin-deficient ob/ob mice treated with a high-fat diet compared to that in control lean mice. RNA sequencing analysis demonstrates that the levels of endothelial guidance molecules such as Roundabout proteins (ROBO/SLIT), which contribute to angiogenesis, are significantly higher in post-PNX obese mouse lungs compared to those from lean mouse lungs. The mRNA levels of Robo1, 2, and 4 and Slit2 are higher in the mouse lung tissues collected from post-PNX obese mice compared to those from lean mouse lungs when analyzed using quantitative RT-PCR. Network analysis reveals that the genes listed in the top 20 BP GO terms of cellular responses including cell migration, cell proliferation and extracellular matrix assembly interact with ROBO/SLIT during post-PNX lung growth. These results suggestthat ROBO/SLIT signaling may mediate the inhibition of post-PNX regenerative lung growth in obese mice. ROBO/SLIT may be the key signaling mechanism to restore lung regeneration in obese population.