Salivary gland function, development, and regeneration.

Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury, will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular, and genetic levels: the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis, and regeneration, and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.

Salivary gland development: a template for regeneration.

The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review will focus on research on mouse submandibular gland development and the translation of this basic research toward therapy for patients suffering from salivary hypofunction. Here we review the most recent literature that has enabled a better understanding of the mechanisms of salivary gland development. Additionally, we discuss approaches proposed to restore salivary function using gene and cell-based therapy. Increasing our understanding of the developmental mechanisms involved during development is critical to design effective therapies for regeneration and repair of damaged glands.

Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture.

Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.

Neurotrophin signaling is a central mechanism of salivary dysfunction after irradiation that disrupts myoepithelial cells.

The mechanisms that prevent regeneration of irradiated (IR) salivary glands remain elusive. Bulk RNAseq of IR versus non-IR human salivary glands showed that neurotrophin signaling is highly disrupted post-radiation. Neurotrophin receptors (NTRs) were significantly upregulated in myoepithelial cells (MECs) post-IR, and single cell RNAseq revealed that MECs pericytes, and duct cells are the main sources of neurotrophin ligands. Using two ex vivo models, we show that nerve growth factor (NGF) induces expression of MEC genes during development, and upregulation of NTRs in adult MECs is associated with stress-induced plasticity and morphological abnormalities in IR human glands. As MECs are epithelial progenitors after gland damage and are required for proper acinar cell contraction and secretion, we propose that MEC-specific upregulation of NTRs post-IR disrupts MEC differentiation and potentially impedes the ability of the gland to regenerate.

Increased 3-O-sulfated heparan sulfate in Alzheimer's disease brain is associated with genetic risk gene HS3ST1.

HS3ST1 is a genetic risk gene associated with Alzheimer's disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3-O-sulfated HS displayed sevenfold increase in the AD group (n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3-O-sulfated HS is made by 3-O-sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3-O-sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3-O-sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.

Loss of Hs3st3a1 or Hs3st3b1 enzymes alters heparan sulfate to reduce epithelial morphogenesis and adult salivary gland function.

Heparan sulfate 3-O-sulfotransferases generate highly sulfated but rare 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes but that Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. To analyze their in vivo functions, we generated both Hs3st3a1-/- and Hs3st3b1-/- single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1-/- mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1-/- HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. Understanding how 3-O-sulfation regulates myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration.

CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands.

Salivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis.

Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction.

Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus with recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 hr before irradiation, and compared with a control adenovirus. The control-treated glands have ∼50% reduction in salivary flow 60 days post-irradiation, whereas neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation, but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

Linking data to decision-making: applying qualitative data analysis methods and software to identify mechanisms for using outcomes data.

A multiple case study was conducted to examine how staff in child out-of-home care programs used data from an Outcomes Management System (OMS) and other sources to inform decision-making. Data collection consisted of thirty-seven semi-structured interviews with clinicians, managers, and directors from two treatment foster care programs and two residential treatment centers, and individuals involved with developing the OMS; and observations of clinical and quality management meetings. Case study and grounded theory methodology guided analyses. The application of qualitative data analysis software is described. Results show that although staff rarely used data from the OMS, they did rely on other sources of systematically collected information to inform clinical, quality management, and program decisions. Analyses of how staff used these data suggest that improving the utility of OMS will involve encouraging staff to participate in data-based decision-making, and designing and implementing OMS in a manner that reflects how decision-making processes operate.

The function of heparan sulfate during branching morphogenesis.

Branching morphogenesis is a fundamental process in the development of diverse epithelial organs such as the lung, kidney, liver, pancreas, prostate, salivary, lacrimal and mammary glands. A unifying theme during organogenesis is the importance of epithelial cell interactions with the extracellular matrix (ECM) and growth factors (GFs). The diverse developmental mechanisms giving rise to these epithelial organs involve many organ-specific GFs, but a unifying paradigm during organogenesis is the regulation of GF activity by heparan sulfates (HS) on the cell surface and in the ECM. This primarily involves the interactions of GFs with the sulfated side-chains of HS proteoglycans. HS is one of the most diverse biopolymers and modulates GF binding and signaling at the cell surface and in the ECM of all tissues. Here, we review what is known about how HS regulates branching morphogenesis of epithelial organs with emphasis on the developing salivary gland, which is a classic model to investigate epithelial-ECM interactions. We also address the structure, biosynthesis, turnover and function of HS during organogenesis. Understanding the regulatory mechanisms that control HS dynamics may aid in the development of therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine.

Exosomal MicroRNA Transport from Salivary Mesenchyme Regulates Epithelial Progenitor Expansion during Organogenesis.

Epithelial-mesenchymal interactions involve fundamental communication between tissues during organogenesis and are primarily regulated by growth factors and extracellular matrix. It is unclear whether RNA-containing exosomes are mobile genetic signals regulating epithelial-mesenchymal interactions. Here we identify that exosomes loaded with mesenchyme-specific mature microRNA contribute mobile genetic signals from mesenchyme to epithelium. The mature mesenchymal miR-133b-3p, loaded into exosomes, was transported from mesenchyme to the salivary epithelium, which did not express primary miR-133b-3p. Knockdown of miR-133b-3p in culture decreased endbud morphogenesis, reduced proliferation of epithelial KIT+ progenitors, and increased expression of a target gene, Disco-interacting protein 2 homolog B (Dip2b). DIP2B, which is involved in DNA methylation, was localized with 5-methylcytosine in the prophase nucleus of a subset of KIT+ progenitors during mitosis. In summary, exosomal transport of miR-133b-3p from mesenchyme to epithelium decreases DIP2B, which may function as an epigenetic regulator of genes responsible for KIT+ progenitor expansion during organogenesis.

Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture.

Radiotherapy for head and neck cancer often has undesirable effects on salivary glands that lead to xerostomia or severe dry mouth, which can increase oral infections. Our goal is to engineer functional, three-dimensional (3D) salivary gland neotissue for autologous implantation to provide permanent relief. An immediate need exists to obtain autologous adult progenitor cells as the use of embryonic and induced pluripotent stem cells potentially pose serious risks such as teratogenicity and immunogenic rejection. Here, we report an expandable population of primary salivary human stem/progenitor cells (hS/PCs) that can be reproducibly and scalably isolated and propagated from tissue biopsies. These cells have increased expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) compared with differentiation markers of the parotid gland (acinar: MIST1/BHLHA15 and AMY1A; ductal: K19 and TFCP2L1). Isolated hS/PCs grown in suspension formed primary and secondary spheres and could be maintained in long-term 3D hydrogel culture. When grown in a customized 3D modular hyaluronate-based hydrogel system modified with bioactive basement membrane-derived peptides, levels of progenitor markers, indices of proliferation, and viability of hS/PCs were enhanced. When appropriate microenvironmental cues were provided in a controlled manner in 3D, such as stimulation with ß-adrenergic and cholinergic agonists, hS/PCs differentiated into an acinar-like lineage, needed for saliva production. We conclude that the stem/progenitor potential of adult hS/PCs isolated without antigenic sorting or clonal expansion in suspension, combined with their ability to differentiate into specialized salivary cell lineages in a human-compatible culture system, makes them ideal for use in 3D bioengineered salivary gland applications. Stem Cells Translational Medicine 2017;6:110-120.

Submandibular parasympathetic gangliogenesis requires sprouty-dependent Wnt signals from epithelial progenitors.

Parasympathetic innervation is critical for submandibular gland (SMG) development and regeneration. Parasympathetic ganglia (PSG) are derived from Schwann cell precursors that migrate along nerves, differentiate into neurons, and coalesce within their target tissue to form ganglia. However, signals that initiate gangliogenesis after the precursors differentiate into neurons are unknown. We found that deleting negative regulators of FGF signaling, Sprouty1 and Sprouty2 (Spry1/2DKO), resulted in a striking loss of gangliogenesis, innervation, and keratin 5-positive (K5+) epithelial progenitors in the SMG. Here we identify Wnts produced by K5+ progenitors in the SMG as key mediators of gangliogenesis. Wnt signaling increases survival and proliferation of PSG neurons, and inhibiting Wnt signaling disrupts gangliogenesis and organ innervation. Activating Wnt signaling and reducing FGF gene dosage rescues gangliogenesis and innervation in both the Spry1/2DKO SMG and pancreas. Thus, K5+ progenitors produce Wnt signals to establish the PSG-epithelial communication required for organ innervation and progenitor cell maintenance.

Hs3st3-modified heparan sulfate controls KIT+ progenitor expansion by regulating 3-O-sulfotransferases.

The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.

Consumer support for health information exchange and personal health records: a regional health information organization survey.

In order to characterize consumer support for electronic health information exchange (HIE) and personal health records (PHRs) in a community where HIE is underway, we conducted a survey of English speaking adults who visited primary care practices participating in a regional community-wide clinical data exchange, during August, 2008. Amongst the 117 respondents, a majority supported physicians' use of HIE (83%) or expressed interest in potentially using PHRs (76%). Consumers' comfort sending personal information electronically over the Internet and their perceptions regarding the potential benefits of HIE were independently associated with their support for HIE. Consumers' prior experience using the Internet to manage their healthcare, perceptions regarding the potential benefits of PHRs and college education were independently associated with potential PHR use. Bolstering consumer support for HIE and PHRs will require addressing privacy and security concerns, demonstrating clinical benefits, and reaching out to those who are less educated and computer literate.

Push and pull: physician usage of and satisfaction with health information exchange.

Federal policy toward health information exchange (HIE), the electronic transfer of patient data across organizations, has evolved to support two forms-push, or point-to-point data transmission, and pull, or multisource data aggregation. HIE usage is associated with user satisfaction, but existing quantitative research is limited to settings where only pull HIE is available. To address this gap, we surveyed 99 physicians regarding usage of and satisfaction with push HIE and pull HIE available in their communities as well as effects of HIE on practice and overall HIE satisfaction. In five of nine measures, respondents reported being very satisfied with push HIE more often than pull HIE (p < 0.05). Physicians were at least four times as likely to report being very satisfied with HIE overall if they were pediatricians, were very satisfied with push HIE, or noted that HIE improved their access to complete information. Findings have implications for HIE implementation and policy.

Consumer attitudes toward personal health records in a beacon community.

OBJECTIVE: To characterize consumers' attitudes about personal health records (PHRs), electronic tools that enable consumers to securely access, manage, and share their health information, in a community participating in health information technology initiatives. STUDY DESIGN: Cross-sectional study. METHODS: A random-digit-dial telephone survey about PHRs was conducted among adult residents of New York State's greater Buffalo region. Multivariate regression analyses identified factors associated with potential PHR use. RESULTS: We obtained a 79% (n = 200) response rate. Many respondents (70%) would potentially use PHRs. Consumers wanted PHRs to incorporate an array of information, including immunization records (89%) and providers visited (88%). They expressed interest in several online activities, including accessing their family members' healthcare information (71%). Potential PHR use was associated with perceptions that PHRs would improve privacy and security of medical information (odds ratio [OR] 4.7; 95% confidence interval [CI] 1.1, 20.1), understanding regarding health (OR 3.7; 95% CI 1.3, 11.1), and overall quality of care (OR 3.6; 95% CI 1.2, 10.6). Potential PHR use was associated with annual household income of more than $30,000 (OR 3.9; 95% CI 1.3, 11.9) and experience looking up health information online (OR 3.0; 95% CI 1.1, 8.1). CONCLUSIONS: Consumers expressed great interest in using PHRs and wanted comprehensive PHRs. However, the "digital divide" between those with varying levels of Internet experience and concerns about PHRs' effect on privacy and security of medical information may limit use. Designing PHRs that incorporate consumer preferences and developing policies that address these barriers may increase consumers' PHR use.

Low-income, ethnically diverse consumers' perspective on health information exchange and personal health records.

We surveyed low-income, ethnically diverse consumers regarding their attitudes towards providers' use of electronic health information exchange (HIE) and consumer use of HIE through personal health records (PHRs). Amongst respondents (n=214), 48% had an annual household income below $15,000 and 62% spoke a language other than English at home. A majority indicated that they supported providers' use of HIE (61%). Support for providers' use of HIE was independently associated with consumer willingness to permit health care providers other than their primary care doctor to view their electronic medical record information (odds ratio (OR)=2.92, 95% confidence interval (CI)=1.31-6.50) and beliefs that electronic health record use would improve quality of care (OR=2.70, 95% CI=1.18-6.18). Seventy-eight percent would potentially use PHRs. Potential PHR use was independently associated with Internet usage rates, (OR=4.46, 95% CI=1.77-11.22), belief that PHR use would improve their understanding of their own healthcare (OR=3.12, 95% CI=1.27-7.67) and comfort with sharing PHR data with their primary care doctor (OR=2.79, 95% CI=1.09-7.11). Low-income, ethnically diverse consumers affected by interoperable health information technology (IT) initiatives largely support using PHRs and HIE, provided these systems demonstrate benefits and address the privacy and security of their electronic health information. Although we found interest in PHRs comparable or higher than nationally representative populations, support for HIE was lower, and thus efforts will need to be made to engage low-income and ethnically diverse consumers to participate in interoperable health IT initiatives.

Differential interactions of FGFs with heparan sulfate control gradient formation and branching morphogenesis.

The developmental activities of morphogens depend on the gradients that they form in the extracellular matrix. Here, we show that differences in the binding of fibroblast growth factor 7 (FGF7) and FGF10 to heparan sulfate (HS) underlie the formation of different gradients that dictate distinct activities during branching morphogenesis. Reducing the binding affinity of FGF10 for HS by mutating a single residue in its HS-binding pocket converted FGF10 into a functional mimic of FGF7 with respect to gradient formation and regulation of branching morphogenesis. In particular, the mutant form of FGF10 caused lacrimal and salivary gland epithelium buds to branch rather than to elongate. In contrast, mutations that reduced the affinity of the FGF10 for its receptor affected the extent, but not the nature, of the response. Our data may provide a general model for understanding how binding to HS regulates other morphogenetic gradients.

Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation.

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.