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In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic’s Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618., PKU patients have elevated phenylalanine levels which can result in neurological impairment. Here the authors utilize biosensor-based ultra-high-throughput screening to optimize PAL activity in a synthetic biotic platform for improved in vivo performance.

Characterized by rapid growth and diffusely infiltrative spread, glioblastoma (GBM) is the most common and most malignant primary brain cancer with a very poor prognosis and a standard of care that hasn’t been improved in over 15 years. The need for new therapies that can directly or indirectly target heterogeneous tumor cell populations to inhibit cell migration in addition to proliferation is highlighted by the recent efforts to develop targeted therapies to treat the vast majority of GBM patients whose tumors impede gross-total resection and develop resistance to chemoradiation. Galectin-3, a carbohydrate-binding protein overexpressed in astrocytes, microglia/macrophages, and the stroma of GBM tumors is a potent modulator of cell proliferation, migration, angiogenesis, T-cell inhibition, and M2 macrophage polarization. We and others have confirmed the association of Galectin-3 expression to lower survival in glioma patients. Additionally, tumor Galectin-3 levels have been shown to increase following exposure to temozolomide or radiation alone or in combination, thus more greatly increasing the need for targeting Galectin-3 in GBM patients following initial treatment. Here, we characterize the therapeutic effects of the anti-Galectin-3 antibody (Gal3 Ab) we have developed for the ability to inhibit GBM tumorigenic and metastatic potential in several in vitro assays and in vivo in heterotopic and orthotopic mouse models. Human GBM cells treated with Gal3 Ab show a significant decrease in cell viability, migration, and invasion in vitro. In vivo, the Gal3 Ab decreases tumor burden and provides a survival benefit in combination with temozolomide compared to the current standard-of-care treatment alone. Our studies indicate the potential for our novel anti-Galectin-3 to be added to the very limited arsenal of potential treatments for GBM. Citation Format: ANNE MARIE BARRETTE, Krutika Deshpande, Marvin Cadiente, Anji Bei, Xi Wang, Teodelinda Mirabella, Dongxu Sun. A novel anti-Galectin-3 antibody therapeutic for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1146.

About 43,600 women in the U.S. are expected to die in 2021 from breast cancer. Triple negative breast cancer (TNBC), which accounts for approximately 15% of all breast cancers, is diagnosed by exclusion of expression and/or amplification of three biomarkers (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]).Characterized by a high risk of relapse (one half of patients with early stages experience recurrence), short overall survival and progression-free survival (life expectancy of few months after failure of first-line chemotherapy), TNBC represents a high unmet medical need. Galectin-3 plays multiple roles in tumor initiation and development via a variety of cellular processes, spanning from angiogenesis to tumor migration. Galectin-3 (Gal3) expression is induced in TNBC patients and highly correlates with poor survival, as shown by our and others’ bioinformatics analysis from public database. We recently discovered and patented a series of antibodies with high specificity and affinity for Gal3, one of which is currently tested in human volunteers. This clinical candidate has shown superior pharmacokinetic properties and clean toxicity profile in GLP studies on non-human primates. Here, we show that anti-Gal3 inhibit both anchorage dependent and independent growth of TNBC cells, as well as Gal3-induced escape from NK cell killing. Also, anti-Gal3 proved to be efficacious in in vivo studies, where TNBC cells were implanted in mammary fat pads of mice and tumor progression, measured as growth of primary mass and presence of metastatic nodules in the lungs, was followed during the two months of treatment. The results presented herein support the conclusion that our therapeutic modality could offer an alternative promising treatment of TNBC. Citation Format: Li Zhang, Heng Wu, Karyssa Palopo, Jie Hu, Marvin Cadiente, Anh Nguyen, Teodelinda Mirabella, Dongxu Sun. A novel anti-Galectin-3 antibody therapeutic for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1147.

Angiogenic sprouting is a critical process involved in vascular network formation within tissues. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell–cell junctions, forming patent structures that support blood flow. Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell–cell dissociation primarily between tip and stalk cells. Closer examination of cell–cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell–cell adhesions during collective motility.

Summary The murine embryonic blood-brain barrier (BBB) consists of endothelial cells (ECs), pericytes (PCs), and basement membrane. Although PCs are critical for inducing vascular stability, signaling pathways in PCs that regulate EC morphogenesis during BBB development remain unexplored. Herein, we find that murine embryos lacking the transforming growth factor β (TGF-β) receptor activin receptor-like kinase 5 (Alk5) in brain PCs (mutants) develop gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH). The germinal matrix (GM) is a highly vascularized structure rich in neuronal and glial precursors. We show that GM microvessels of mutants display abnormal dilation, reduced PC coverage, EC hyperproliferation, reduced basement membrane collagen, and enhanced perivascular matrix metalloproteinase activity. Furthermore, ALK5-depleted PCs downregulate tissue inhibitor of matrix metalloproteinase 3 (TIMP3), and TIMP3 administration to mutants improves endothelial morphogenesis and attenuates GMH-IVH. Overall, our findings reveal a key role for PC ALK5 in regulating brain endothelial morphogenesis and a substantial therapeutic potential for TIMP3 during GMH-IVH.

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that

In spite of the vast collective experience in tissue engineering, control of both tissue architecture and scale are fundamental translational roadblocks. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. Here, we introduce an approach called ‘SEEDs’ (‘in Situ Expansion of Engineered Devices’), in which we fabricate a structurally organized engineered tissue unit that expands in response to regenerative cues after implantation. We find that tissues containing pre-patterned human primary hepatocytes, endothelial cells, and stromal cells in degradable hydrogel expand over 50-fold over the course of 11 weeks in animals with liver injury, with concomitant increased function as characterized by the production of multiple human liver proteins. Histologically, we observe the emergence of stereotypical microstructure via coordinated growth of hepatocytes in close juxtaposition with a perfused, chimeric vasculature. Importantly, we demonstrate the utility of this platform for probing the impact of multicellular geometric architecture on tissue expansion in response to regenerative cues. This approach represents a hybrid strategy that harnesses both biology and engineering to deploy a limited cell mass more efficiently than either approach could do in isolation, and thus offers a new convergent paradigm for tissue engineering.

Objective: Angiogenesis is crucial to maintain tissue homeostasis under nutrient and oxygen deprivation (ischemia). Although considerable evidence supports that angiogenesis is regulated by hypoxia-HIF1α induction of vascular endothelial growth factor (VEGF), the role of nutrient deprivation in angiogenesis is poorly defined. Approach and Results: We report that nutrient deprivation in the form of dietary sulfur amino acid restriction (Methionine/cysteine Restriction; MR) promotes VEGF expression and functional growth of new capillaries in skeletal muscle of mice (Fig.1 A, B). This occurred independently of hypoxia or HIF1α, but instead required the amino acid-sensing eIF2α kinase GCN2 and the transcription factor ATF4 (Fig. 1C). In addition to increased VEGF, nutrient deprivation boosted production of the pro-angiogenic gas hydrogen sulfide (H 2 S) via increased GCN2/ATF4-dependent expression of the H 2 S-generating enzyme cystathionine-gamma-lyase (CGL). The genetic requirement for CGL in angiogenesis triggered by nutrient deprivation, exercise or local VEGF overexpression, as well as the ability of local CGL overexpression to promote angiogenesis in vivo, revealed the critical importance of CGL-derived H 2 S in angiogenesis (Fig. 1D). Finally, plasma H 2 S was reduced in patients with vascular disease (versus non-diseased age-matched controls), and correlated with 2-year survival following vascular surgery (Fig.1 E, F). Conclusions: These data reveal a nutrient-sensing pathway targetable by diet as a previously unrecognized central regulator of VEGF expression and angiogenesis independent of canonical hypoxic signaling. This discovery points to novel dietary interventions and GCN2/ATF4/CGL/H 2 S-based strategies to manipulate angiogenesis. Figure 1

The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host's progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine.

Angiogenesis, the formation of new blood vessels by endothelial cells (EC), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in EC in vitro, and increased capillary density in mouse skeletal muscle in vivo, via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H(2)S) production. H(2)S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

The repair of skeletal defects remains a substantial economic and biomedical burden. The extra-embryonic fetal stem cells derived from amniotic fluid (AFSCs) have been used for the treatment of large bone defects, but mechanisms of repair are not clear. Here we studied the potential contribution of human AFSCs to the modeling of an ectopic bone. We found that AFSCs are not osteogenic in vivo, and, compared to bone marrow-derived stromal cells, recruit more host CD31 and VEGF-R 2 positive cells. Finally, when AFSCs were co-implanted with human-bone forming cells, a normo-osteosynthesis occurred, the engineered ossicle was hyper-vascularized, but AFSCs were not retrieved in the implant within 2 weeks. We concluded that AFSCs do not contribute to the deposition of new bone, but act as a powerful proinflammatory/proangiogenic boost, driving a host response, ending in AFSC clearance and vascularization of the bone environment. In our model, a source of osteocommitted cells, capable to engraft and proliferate in vivo, is needed in order to engineer bone. The angio-attractant properties of AFSCs could be exploited in strategies of endogenous cell homing to actively recruit host progenitors into a predefined anatomic location for in situ bone tissue regeneration.

Erratum to “Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model” [Biomaterials 32 (2011) 3689e3699] Teodelinda Mirabella *, Michele Cilli , Sebastiano Carlone , Ranieri Cancedda , Chiara Gentili a,b Department of Oncology, Biology and Genetics, University of Genoa, Largo Rosanna Benzi 10, 16132 Genova, Italy National Cancer Research Institute, Largo Rosana Benzi 10, 16132 Genova, Italy