Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.     

Microvascular submandibular gland transfer for severe cases of keratoconjunctivitis sicca

Free submandibular salivary gland transfer was investigated as a surgical method for the treatment of severe keratoconjunctivitis sicca. In an animal model, we examined the tolerance of warm ischemia of the submandibular gland. After temporary interruption of the blood supply (1 to 6 hours), the morphologic changes in the submandibular gland were analyzed histologically and immunohistochemically in 41 rabbits. From 1.5 hours ischemia onward, an increasing structural damage of the parenchyma with emphasis on the secretory cells was seen. Six hours of ischemia caused total necrosis of the salivary gland. Our clinical experience includes 24 highly selected patients suffering from keratoconjunctivitis sicca, in whom we transferred 31 autologous submandibular glands to the temple for permanent autologous tear substitution within the past 4 years. The glands were implanted into a pocket prepared in the temporalis muscle, and the nourishing vessels were anastomosed to the superficial temporal artery and vein. The submandibular duct was implanted into the upper lateral conjunctival fornix. The transferred glands were left denervated. In addition to the clinical examination, scintigraphy with Tc 99m pertechnetate was used to document the graft's viability after the transfer. Viable incorporation with longstanding secretory function occurred in 26 of the 30 transplanted denervated salivary glands. The resulting lubrication of the treated eyes was irregular for up to 3 months in almost even case. One year after surgery, all patients with a viable transplant developed at least occasional epiphora, which was surgically managed by reducing the size of the graft in 10 patients. No severe side effects were seen in this series. The ophthalmologic evaluation of the method included the assessment of dry eye symptoms and of the volume and quality of ocular lubrication (Schirmer test, fluorescein break-up time), the pathology of the ocular surface (rose bengal staining), and the need for pharmaceutical tear substitutes. One year after surgery, 18 of 27 cases assessed were judged as significantly improved by these tests.     

Regeneration potential of the submandibular gland after transplantation: An immunohistological and scintigraphic study

The aim of this study was to evaluate the regeneration potential of the submandibular gland and the associated morphological and functional impairment after a transient warm ischemia as a result of submandibular gland transfer in humans. 42 rabbits were used for the study. After 1.5 h of transient ischemia, submandibular glands were studied histologically in the following 14 days, one month, two and four months in 32 rabbits. Additionally, the glands were functionally evaluated after one and four months in eleven of these rabbits. Ten rabbits were used to establish the scintigraphic method. In the evaluation of the results unpublished data derived from a previous study by our group has also been considered. The Ki-67 index showed a significant increase of the proliferating rate reaching a peak after 14 days (p = 0.006) and still evident after four months involving mostly the terminal ductal system. There was only a slight increase in the tracer uptake of ischemic glands after one month and a more pronounced one after four months.It was concluded that initial loss of acinar cells by 1.5 h of ischemia released a regeneration process which stabilised after 14 days but continued throughout the whole time of the study. These results correlate with clinical observation in patients with submandibular gland transfer for the treatment of keratoconjunctivitis sicca and explain the alternating secretory function observed within the four months following surgery. Scintigraphy has been found to play a limited role in the evaluation of the regeneration process.     

Microvascular transplantation of the rat submandibular gland

Xerostomia results from salivary gland irradiation during treatment of head and neck malignancies. In addition to having difficulty with speech and swallowing, these patients experience loss of taste, dental caries, and chronic fungal infections. The paired submandibular glands provide 70 percent of the normal salivary flow and are difficult to shield during radiation therapy. Another sicca condition, xerophthalmia, may result from facial nerve injury or other medical disorders and results in pain, corneal ulceration, and possible vision loss. Treatment options for xerostomia are limited, and management of xerophthalmia usually focuses on the eyelids, rather than the fundamental problem of inadequate secretory protection. In this study, a rat model for submandibular gland microvascular transplantation was developed to assess the feasibility of salivary tissue transfer. Sixteen rats underwent submandibular gland transplantation from the neck to the groin. Fourteen of these rats underwent microvascular anastomosis of the vascular pedicle. Ten glands were assessed for viability at 4 days after transplantation, and four glands were examined after 7, 10, 14, or 21 days. By gross and histologic examination, 93 percent of transplanted glands showed expected long-term viability after at least 4 postoperative days. Microvascular techniques were shown to be applicable to the transplantation of submandibular gland salivary tissue. This has not previously been shown in a rat model. It is possible that submandibular glands could be transplanted to the eye for treatment of xerophthalmia and out of the neck during irradiation of the head and neck, with subsequent replantation after treatment as a means of preventing permanent xerostomia.     

Decreased saliva secretion and down-regulation of AQP5 in submandibular gland in irradiated rats

The molecular mechanisms of radiation-induced xerostomia remain unclear. The purpose of this study was to investigate the alterations of aquaporins (AQPs) and Na(+)/K(+)-ATPase in irradiated rat submandibular glands and to test the hypothesis that down-regulation of AQP5 expression in irradiated salivary glands is one of the mechanisms of radiation-induced xerostomia. Saliva from control and irradiated rat submandibular glands was analyzed. The mRNA level of AQP5 in the submandibular glands was assessed by semi-quantitative RT-PCR and in situ hybridization. The protein expression of AQP5, AQP1 and Na(+)/K(+)-ATPase was determined by Western blotting and immunohistochemistry. The body weight, submandibular gland weight, and saliva secretion of irradiated rats significantly decreased by 12, 24 and 32% on day 3 and 24, 16 and 38% on day 30 postirradiation, respectively. There was a significant increase in the protein concentration and osmolality of saliva in irradiated rats on days 3 and 30 postirradiation. However, there was no significant difference between irradiated and control rats in total saliva protein secretion. RT-PCR analysis showed that mRNA expression of AQP5 was significantly down-regulated by 37 and 51% in irradiated rats on days 3 and 30 postirradiation, respectively. Immunoblotting showed that the AQP5 protein level was decreased by 40 and 60% in irradiated glands, in contrast to the slight reductions of AQP1 and Na(+)/K(+)-ATPase proteins. Immunohistochemical analysis demonstrated that loss of AQP5 protein occurred throughout the irradiated glands, while no significant reduction was detected in AQP1 and Na(+)/ K(+)-ATPase labeling density. These results suggest that the preferential down-regulation of AQP5 with minor effects on AQP1 and Na(+)/K(+)-ATPase may contribute to radiation-induced salivary dysfunction.     

Proteomic analysis of human transplanted submandibular gland in patients with epiphora after transplantation

Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner.     

Proteins Identified from Saliva and Salivary Glands of the Chinese Gall Aphid Schlechtendalia chinensis

Aphid saliva plays an essential role in the interaction between aphids and their host plants. Several aphid salivary proteins have been identified but none from galling aphids. Here the salivary proteins from the Chinese gall aphid are analyzed, Schlechtendalia chinensis, via an LC‐MS/MS analysis. A total of 31 proteins are identified directly from saliva collected via an artificial diet, and 141 proteins are identified from extracts derived from dissected salivary glands. Among these identified proteins, 17 are found in both collected saliva and dissected salivary glands. In comparison with salivary proteins from ten other free‐living Hemipterans, the most striking feature of the salivary protein from S. chinensis is the existence of high proportion of proteins with binding activity, including DNA‐, protein‐, ATP‐, and iron‐binding proteins. These proteins maybe involved in gall formation. These results provide a framework for future research to elucidate the molecular basis for gall induction by galling aphids.     

Elafin expression in human fetal and adult submandibular glands

Elafin, a bifunctional protein, has the NH(2)-terminal domain functions as a transglutaminase substrate for crosslinking to lysine-containing proteins and the COOH-terminal whey acidic protein domain as a potent anti-elastase. Human fetal submandibular glands (n=100) and adult submandibular glands (n=10) were used to elucidate the expression pattern of elafin in the developmental processes of human submandibular gland by immunohistochemistry, in situ hybridization, and western blot analysis. Elafin mRNA was expressed both in the gland epithelium and intralobular mesenchymal tissue of fetal submandibular gland in an early developmental stage (10-18 weeks) and an early intermediate developmental stage (EIDS; 19-24 weeks). The elafin antigen was also found in the intralobular mesenchyme of submandibular gland in the same stages. Thereafter, elafin was transitionally expressed in the ducts and acini of submandibular glands. In the late intermediate developmental stage (LIDS; 25-32 weeks) and the late developmental stage (LDS; 33-40 weeks), elafin became markedly positive in the excretory and striated ducts but weakly positive in the intralobular mesenchymal tissue. The elafin was heavily present in the excretory and striated ducts of adult submandibular gland, while it was sparse in the intralobular mesenchymal tissues. Western blot analysis showed the protein extracts from submandibular glands of EIDS, LIDS, and LDS, adult submandibular gland, fetal tissues (8 weeks), and adult parotid saliva migrated into multiple bands, about 25, 50, 65, and 140 kDa, which were higher than the putative size of elafin protein, 12 kDa. These data suggest that elafin, anti-elastase, is an essential component highly utilized during the morphogenetic processes of fetal salivary gland development and continuously plays a role for the protection of the tubuloalveolar structures of adult salivary gland.     

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.     

Effects of saliva from chronically reserpinized rat on Na and K transport in perfused main excretory duct of submandibular gland of normal rat

Reserpine (RES) (0.5 mg/kg body wt, ip) was administered to rats for 7 days. On Day 8 saliva was evoked from these animals by intraperitoneal injection of pilocarpine nitrate (10 mg/kg body wt) and saliva from submandibular and parotid glands was collected separately. These collected salivas were used to perfuse through the main ducts of the submandibular glands of normal rats. After a control period of perfusion of the main duct with bicarbonate saline solution, parotid saliva from RES rats was perfused through the duct followed by regular perfusion. There was inhibition of Na absorption (22%) and K secretion (23%). Moreover, when submandibular saliva from treated rat was perfused through the main duct prior to regular perfusion, there was a decrease in Na absorption (31%) and K secretion (28%). In contrast, perfusion of the main duct with either parotid or submandibular saliva from normal rats caused no significant changes in Na and K transport. The present experiments confirm previous studies that there is some Na-inhibitory factor(s) present in saliva of the chronically RES-treated rat.     

Human parotid and submandibular glands express and secrete surfactant proteins A,B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.     

Sorting of growth hormone-erythropoietin fusion proteins in rat salivary glands

Neuroendocrine and exocrine cells secrete proteins in either a constitutive manner or via the regulated secretory pathway (RSP), but the specific sorting mechanisms involved are not fully understood. After gene transfer to rat salivary glands, the transgenic model proteins human growth hormone (hGH) and erythropoietin (hEpo) are secreted primarily into saliva (RSP; exocrine) and serum (constitutive; endocrine), respectively. We hypothesized that fusion of hGH at either the C-terminus or the N-terminus of hEpo would re-direct hEpo from the bloodstream into saliva. We constructed and expressed two fusion proteins, hEpo-hGH and hGH-hEpo, using serotype 5-adenoviral vectors, and delivered them to rat submandibular glands in vivo via retroductal cannulation. Both the hEpo-hGH and hGH-hEpo fusion proteins, but not hEpo alone, were secreted primarily into saliva (p < 0.0001 and p = 0.0083, respectively). These in vivo studies demonstrate for the first time that hGH, in an N- as well as C-terminal position, influences the secretion of a constitutive pathway protein.     

Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation

Treatment for most patients with head and neck cancers includes ionizing radiation. A consequence of this treatment is irreversible damage to salivary glands (SGs), which is accompanied by a loss of fluid-secreting acinar-cells and a considerable decrease of saliva output. While there are currently no adequate conventional treatments for this condition, cell-based therapies are receiving increasing attention to regenerate SGs. In this study, we investigated whether bone marrow-derived cells (BMDCs) can differentiate into salivary epithelial cells and restore SG function in head and neck irradiated mice. BMDCs from male mice were transplanted into the tail-vein of 18Gy-irradiated female mice. Salivary output was increased in mice that received BMDCs transplantation at week 8 and 24 post-irradiation. At 24 weeks after irradiation (IR), harvested SGs (submandibular and parotid glands) of BMDC-treated mice had greater weights than those of non-treated mice. Histological analysis shows that SGs of treated mice demonstrated an increased level of tissue regenerative activity such as blood vessel formation and cell proliferation, while apoptotic activity was increased in non-transplanted mice. The expression of stem cell markers (Sca-1 or c-kit) was detected in BMDC-treated SGs. Finally, we detected an increased ratio of acinar-cell area and approximately 9% of Y-chromosome-positive (donor-derived) salivary epithelial cells in BMDC-treated mice. We propose here that cell therapy using BMDCs can rescue the functional damage of irradiated SGs by direct differentiation of donor BMDCs into salivary epithelial cells.     

Altered fractalkine cleavage potentially promotes local inflammation in NOD salivary gland

Introduction

In the nonobese diabetic (NOD) mouse model of Sjögren's syndrome, lymphocytic infiltration is preceded by an accumulation of dendritic cells in the submandibular glands (SMGs). NOD mice also exhibit an increased frequency of mature, fractalkine receptor (CX3C chemokine receptor [CX3CR]1) expressing monocytes, which are considered to be precursors for tissue dendritic cells. To unravel further the role played by fractalkine-CX3CR1 interactions in the salivary gland inflammation, we studied the expression of fractalkine in NOD SMGs.

Methods

We studied protein expression using Western blot analysis of whole tissue lysates. Protease activity was measured in salivary gland tissue lysates using fluorimetric substrates. Digestive capacity of enzymes was determined by in vitro incubation of recombinant enzyme and fractalkine, followed by protein staining and Western blot.

Results

Fractalkine was detected in salivary glands of both NOD and control mice at all ages. Western blot analysis showed fractalkine cleavage with increasing age, which was more pronounced in NOD mice. This cleavage resulted in a decrease in the 31 kDa form of the protein, and the generation of an approximately 19 kDa band. Furthermore, in NOD animals older than 15 weeks, we noted the presence of a unique approximately 17 kDa fragment. This cleavage was organ specific, because it did not occur in brain or pancreas. Increased gelatinase and α-secretase activity were detected in NOD SMG and contributed to cleavage of the 31 kDa protein. Because aberrant cleavage products may induce autoimmunity, we studied the presence of autoantibodies against fractalkine. Indeed, NOD mice exhibited significantly more antibodies against fractalkine than did control animals.

Conclusion

These data indicate that aberrant proteolytic activity in the NOD SMG results in increased fractalkine cleavage and generation of a unique fractalkine fragment. This specific cleavage may contribute to autoimmunity.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

Phenylephrine protects autotransplanted rabbit submandibular gland from apoptosis

Submandibular gland (SMG) autotransplantation is an effective treatment for severe keratoconjunctivitis sicca. Our previous studies have shown that phenylephrine attenuates structural injury and promotes cell proliferation in autotransplanted rabbit SMG. However, the mechanism by which phenylephrine reduces the injury has not been fully evaluated. In this study, we investigate the ability of phenylephrine to inhibit apoptosis in autotransplanted rabbit SMG. We observed that apoptosis occurred in the early phase of SMG transplantation and that phenylephrine treatment protected transplanted SMG from apoptosis. Furthermore, we found that phenylephrine could significantly upregulate the expression of Bcl-2, downregulate the expression of Bax, and inhibit the activation of both caspase-3 and p38 mitogen-activated protein kinase in autotransplanted SMG. Therefore, the cytoprotective effects of phenylephrine on autotransplanted SMG may be a novel clinical strategy for autotransplanted SMG protection during the early postoperative stage of transplantation.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family

Mouse submandibular gland prorenin-converting enzyme (PRECE) consists of the two polypeptide chains of 17 and 10 kDa and cleaves mouse Ren-2 prorenin at a dibasic site to yield mature renin. Western blot analysis using an antiserum against this enzyme gave rise to multiple bands in mouse submandibular glands, suggesting that PRECE is a member of a protease family. Partial amino acid sequence analysis of purified PRECE and cloning and sequence analyses of its cDNA indicated that it is identical to the mGK-13 gene product, epidermal growth factor-binding protein type B, which is a member of the glandular kallikrein family and is involved in maturation of epidermal growth factor. Conditioned medium from Chinese hamster ovary cells transfected with an expression plasmid for PRECE had prorenin converting activity. These results indicate that PRECE is involved in the maturation of two bioactive polypeptides expressed in mouse submandibular glands, Ren-2 renin and epidermal growth factor.