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Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in ...regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.
Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.
Polyarteritis nodosa is a systemic necrotizing vasculitis with a pathogenesis that is poorly understood. We identified six families with multiple cases of systemic and cutaneous polyarteritis nodosa, ...consistent with autosomal recessive inheritance. In most cases, onset of the disease occurred during childhood.
We carried out exome sequencing in persons from multiply affected families of Georgian Jewish or German ancestry. We performed targeted sequencing in additional family members and in unrelated affected persons, 3 of Georgian Jewish ancestry and 14 of Turkish ancestry. Mutations were assessed by testing their effect on enzymatic activity in serum specimens from patients, analysis of protein structure, expression in mammalian cells, and biophysical analysis of purified protein.
In all the families, vasculitis was caused by recessive mutations in CECR1, the gene encoding adenosine deaminase 2 (ADA2). All the Georgian Jewish patients were homozygous for a mutation encoding a Gly47Arg substitution, the German patients were compound heterozygous for Arg169Gln and Pro251Leu mutations, and one Turkish patient was compound heterozygous for Gly47Val and Trp264Ser mutations. In the endogamous Georgian Jewish population, the Gly47Arg carrier frequency was 0.102, which is consistent with the high prevalence of disease. The other mutations either were found in only one family member or patient or were extremely rare. ADA2 activity was significantly reduced in serum specimens from patients. Expression in human embryonic kidney 293T cells revealed low amounts of mutant secreted protein.
Recessive loss-of-function mutations of ADA2, a growth factor that is the major extracellular adenosine deaminase, can cause polyarteritis nodosa vasculopathy with highly varied clinical expression. (Funded by the Shaare Zedek Medical Center and others.).
Deficiency of adenosine deaminase 2 (DADA2) is caused by biallelic deleterious mutations in CECR1. DADA2 results in variable autoinflammation and vasculopathy (recurrent fevers, livedo reticularis, ...polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency and bone marrow failure. Tumor necrosis factor-α blockade is the treatment of choice for the autoinflammation and vascular manifestations. Hematopoietic stem cell transplantation (HSCT) represents a potential definitive treatment. We present a cohort of 14 patients from 6 countries who received HSCT for DADA2. Indication for HSCT was bone marrow dysfunction or immunodeficiency. Six of 14 patients had vasculitis pre-HSCT. The median age at HSCT was 7.5 years. Conditioning regimens were myeloablative (9) and reduced intensity (5). Donors were HLA-matched sibling (n = 1), HLA-matched unrelated (n = 9), HLA-mismatched unrelated (n = 3), and HLA haploidentical sibling (n = 1). All patients are alive and well with no new vascular events and resolution of hematological and immunological phenotype at a median follow-up of 18 months (range, 5 months to 13 years). Plasma ADA2 enzyme activity normalized in those tested post-HSCT (7/7), as early as day +14 (myeloid engraftment). Post-HSCT hematological autoimmunity (cytopenias) was reported in 4 patients, acute graft-versus-host disease grade 1 in 2, grade 2 in 3, and grade 3-4 in 1, and moderate chronic graft-versus-host disease in 1 patient. In conclusion, in 14 patients, HSCT was an effective and definitive treatment of DADA2.
•HSCT represents an effective and definitive treatment of DADA2.•HSCT can cure the immunological, hematological, and vascular phenotype of DADA2 with 100% survival at median follow-up of 18 months.
Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a ...CD49a+Eomes+ subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a+Eomes+ NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.
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•Uterine CD49a+Eomes+ trNK cell subset secretes GPFs including pleiotrophin and osteoglycin•A decrease in the GPF-secreting trNK cell subset impairs fetal development•Adoptive transfer of uterine-like trNK cells can reverse impaired fetal growth
Maternal nourishment of the fetus at early stage has remained undefined. Fu et al. identify CD49a+Eomes+ trNK cells in the uterus that secrete growth-promoting factors enhancing fetal growth during critical early stages of fetal development.
Cells of the osteoblast lineage affect the homing and the number of long-term repopulating haematopoietic stem cells, haematopoietic stem cell mobilization and lineage determination and B cell ...lymphopoiesis. Osteoblasts were recently implicated in pre-leukaemic conditions in mice. However, a single genetic change in osteoblasts that can induce leukaemogenesis has not been shown. Here we show that an activating mutation of β-catenin in mouse osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukaemia with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand jagged 1 in osteoblasts. Subsequent activation of Notch signalling in haematopoietic stem cell progenitors induces the malignant changes. Genetic or pharmacological inhibition of Notch signalling ameliorates acute myeloid leukaemia and demonstrates the pathogenic role of the Notch pathway. In 38% of patients with myelodysplastic syndromes or acute myeloid leukaemia, increased β-catenin signalling and nuclear accumulation was identified in osteoblasts and these patients showed increased Notch signalling in haematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce acute myeloid leukaemia, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to acute myeloid leukaemia.
Growth factors (GFs) are critical in tissue repair, but their translation to clinical use has been modest. Physiologically, GF interactions with extracellular matrix (ECM) components facilitate ...localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in their clinically used forms could underlie the limited translation. We discovered that a domain in placenta growth factor-2 (PlGF-2123-144) binds exceptionally strongly and promiscuously to ECM proteins. By fusing this domain to the GFs vascular endothelial growth factor–A, platelet-derived growth factor–BB, and bone morphogenetic protein–2, we generated engineered GF variants with super-affinity to the ECM. These ECM super-affinity GFs induced repair in rodent models of chronic wounds and bone defects that was greatly enhanced as compared to treatment with the wild-type GFs, demonstrating that this approach may be useful in several regenerative medicine applications.
The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as ...phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.
Discovery of the growth factor activity of ADA2.
ADAs play a pivotal role in regulating the level of adenosine, a signaling molecule controlling a variety of cellular responses by binding to and ...activating four ADRs. Two enzymes, ADA1 and ADA2, are known to possess ADA activity in humans. Although the structure of ADA1 and its role in lymphocytic activation have been known for a long time, the structure and function of ADA2, a member of ADGF, remain enigmatic. Here, we found that ADA2 is secreted by monocytes undergoing differentiation into macrophages or DCs and that it binds to the cell surface via proteoglycans and ADRs. We demonstrate that ADA1 and ADA2 increase the rate of proliferation of monocyte‐activated CD4+ T cells independently of their catalytic activity. We also show that ADA2 induces T cell‐dependent differentiation of monocytes into macrophages and stimulates macrophage proliferation. Our discovery of the growth factor‐like activity of ADA2 explains clinical observations and suggests that this enzyme could be used as a drug candidate to modulate the immune responses during inflammation and cancer.
Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a ...matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvβ5/αvβ3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvβ5/αvβ3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvβ5/αvβ3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvβ5/NF-κB/JAG1 as a critical axis for biliary injury repair.
Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by deleterious ADA2 variants. The frequency of these variants in the general population, and hence the expected ...disease prevalence, remain unknown.
We aimed to characterize the functional impact and carrier frequency of ADA2 variants.
We performed functional studies and in silico analysis on 163 ADA2 variants, including DADA2-associated variants and population variants identified in the Genome Aggregation Database. We estimated the carrier rate using the aggregate frequency of deleterious variants.
Functional studies of ADA2 variants revealed that 77 (91%) of 85 of DADA2-associated variants reduced ADA2 enzymatic function by >75%. Analysis of 100 ADA2 variants in the database showed a full spectrum of impact on ADA2 function, rather than a dichotomy of benign versus deleterious variants. We found several in silico algorithms that effectively predicted the impact of ADA2 variants with high sensitivity and specificity, and confirmed a correlation between the residual function of ADA2 variants in vitro and the plasma ADA2 activity of individuals carrying these variants (n = 45; r = 0.649; P < .0001). Using <25% residual enzymatic activity as the cutoff to define potential pathogenicity, integration of our results with the database population data revealed an estimated carrier frequency of at least 1 in 236 individuals, corresponding to an expected DADA2 disease prevalence of ~1 in 222,000 individuals.
Functional annotation guides the interpretation of ADA2 variants to create a framework that enables estimation of DADA2 carrier frequency and disease prevalence.
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