Lymphatic Density Research Articles

Blood microvasculature and lymphatic densities in endometrial polyps and adjacent and distant endometrium.

Objective:Endometrial polyps are localised growths of endometrial tissue containing glands, stroma and blood vessels, covered with epithelium. The reported prevalence of endometrial polyps is dependent upon the population being studied and the uterine imaging technique utilised. The light microscopy literature provides very little information regarding their microvasculature and lymphatic systems; however, a plethora of ultrasound data demonstrating single central arteries in most medium- or large-sized endometrial polyps are well documented.Methods:Archived formalin-fixed paraffin-embedded blocks of endometrial curettings were retrieved from files for women with confirmed endometrial polyps (n = 20) and women with normal endometrium (control endometrium; n = 32). Immunohistochemistry was performed with the antibodies CD31 (blood vessels) and D2-40 (lymphatics). Blood vessels and lymphatics were quantified in endometrial polyps and adjacent, distant and control endometrium.Results:CD31 and D2-40 staining was present in all specimens, although there were no significant differences in blood vessel (F(3,70) = 2.36, p = 0.079) and lymphatic (F(3,70) = 0.16, p = 0.920) densities between endometrial polyps as well as adjacent, distant and control endometrium. There were also no significant differences in women with endometrial polyp-associated bleeding and those with no bleeding. In relation to infertility, there were no significant differences found in blood and lymphatic densities between women with endometrial polyps who were infertile and those with endometrial polyps who were fertile.Conclusion:Small blood vessel wall and perivascular structures rather than the distribution of vessels may be associated with abnormal bleeding.

Regression of Lingual Lymphatic Vessels in Sodium-restricted Mice.

Lymphatic vessel networks can expand and regress, with consequences for interstitial fluid drainage and nutrient supply to tissues, inflammation, and tumor spread. A diet high in sodium stimulates hyperplasia of cutaneous lymphatic capillaries. We hypothesized that dietary sodium restriction would have the opposite effect, shrinking lymphatic capillaries in the tongue. Lingual lymphatic capillary density and size was significantly reduced in mice fed a low-sodium diet (0.03%) for 3 weeks compared with control-fed mice. Blood vessel density was unchanged. Despite lymphatic capillary shrinkage, lingual edema was not observed. The effect on lymphatic capillaries was reversible, as lymphatic density and size in the tongue were restored by 3 weeks on a control diet. Lymphatic hyperplasia induced by a high-sodium diet is dependent on infiltrating macrophages. However, lingual CD68+ macrophage density was unchanged by sodium deficiency, indicating that distinct mechanisms may mediate lymphatic regression. Further studies are needed to test whether dietary sodium restriction is an effective, non-invasive co-therapy for oral cancer.

VEGF-C-mediated cardiac lymphangiogenesis in high salt intake accelerated progression of left ventricular remodeling in spontaneously hypertensive rats

ABSTRACTHigh salt (HS) diet can accelerate the progress of hypertensive left ventricular (LV) remodeling. But the detailed mechanism remains poorly understood. We hypothesized HS intake could impact cardiac lymphangiogenesis through tonicity-responsive enhancer binding protein (TonEBP)/vascular endothelial growth factor-C (VEGF-C) signaling pathway which might play an important role in HS intake accelerated LV remodeling. Eight-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were randomized to 0.5% NaCl (Low salt, LS) and 8% NaCl (high salt, HS) diets for 12 weeks. LV remodeling was determined by echocardiography. LV invasive hemodynamic analysis and morphologic staining (cardiomyocyte hypertrophy, collagen deposition, TonEBP expression, macrophage infiltration and lymphatic density) were performed at the time of sacrifice. The blood pressure of SHR-HS group was significantly increased compared to SHR-LS and WKY groups. Meanwhile, The LV chamber size was markedly enlargement, LV function apparently compromised accompanied with a severe macrophage infiltration, and fibrosis in the perivascular and interstitium of LV compared with SHR-LS group. Furthermore, the expression levels of VEGF-C, TonEBP, and lymphatic markers in SHR-HS group were significantly increased parallel with apparent lymphangiogenesis compared with SHR-LS group. Our work indicates that TonEBP/VEGF-C signaling pathway was up-regulated in HS intake accelerated hypertensive LV remodeling process that may be valuable for further investigation.

Triple negative breast cancer: the kiss of death.

One of the most controversial women malignancies, triple negative breast cancers (TNBCs) are critically overviewed here, being focused on data useful in clinical practice or to improve the therapy and patients survival. TNBCs “choose” young women and its “kiss” is, unfortunately deadly in most cases. Currently, few sparse data are available in literature concerning the origins of TNBC. Vasculogenic mimicry detected in TNBCs, seems to be determined by a population of CD133+ cells and may be stimulated by different pharmacological agents such sunitinib. Despite the fact that TNBCs do not usually metastasize through the lymphatic pathways, TNBCs may be characterized by lymphatic invasion and by an increased lymphatic microvascular density. If TNBCs treatment depends on the molecular profile of the tumor, the same statement may be postulated for TNBCs metastasis. Whether metastases have a similar phenotype as the primary tumor remains an enigma. Therefore, the question: ‘Could TNBC be subject to a standardized, unanimously accepted therapeutic strategy or is it strictly subclass-dependent?’ remains to be further investigated.

Adipose Tissue VEGF‐D Overexpression Increases Lymphatic Vessel Density and Protects Against Inflammation and Insulin Resistance in Obesity

Lymphatic vessels modulate tissue fluid balance and inflammation and provide a conduit for endocrine and lipid transport: all mechanisms that could potentially regulate adipose tissue physiology in obesity. The growth of new lymphatic vessels in is mediated through vascular endothelial growth factor receptor‐3 (VEGFR‐3) signaling. We took advantage of the unique binding of murine VEGF‐D to VEGFR‐3 and generated mice capable of inducible, tissue‐specific expression of murine VEGF‐D under a tightly‐controlled tetracycline response element (TRE) promoter to stimulate lymphangiogenesis. Crossed with adipocyte‐specific adiponectin‐rtTA mice (Adipo‐VD), VEGF‐D overexpression by adipocytes induced de novo lymphangiogenesis in murine white adipose tissue and a massive expansion of brown adipose tissue lymphatics. Upon removal of the doxycycline stimulus, VEGF‐D expression returned to normal and the expanded adipose tissue lymphatics regressed. The new white adipose lymphatic network provided a route for increased glycerol flux during lipolysis. As a chemokine, VEGF‐D expression recruited macrophages and caused fibrosis in white adipose tissue on chow diet, but no negative systemic metabolic consequences were identified. On high fat diet feeding, Adipo‐VD gained equivalent weight and adiposity as littermate controls, but were markedly more insulin sensitive. White adipose tissues in Adipo‐VD mice were full of lymphatics, but exhibited less fibrosis, contained fewer crown‐like structure macrophages, and displayed increased UCP‐1 RNA and protein. Infusion of CD45.1+ leukocytes into CD45.2+ Adipo‐VD mice identified fewer immune cells accumulating in the Adipo‐VD subcutaneous adipose with more trafficking out and into the inguinal lymph node. The increased lymphatic density thus reduces local inflammation in adipose tissue, manifesting as decreased liver triglyceride, increased insulin sensitivity, and overall healthier obesity highlighting the roles of lymphatic in regulating inflammatory pathologies and VEGFR‐3 signaling as a potential target in the metabolic syndrome.

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE−/− mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3+ T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3+ T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.

A Study of D2-40 Immunohistochemical Expression in Colorectal Carcinomas

Background: Colorectal cancer is one of the most common cancers. Factors affecting survival include nodal positivity, lymphovascular invasion (LVI) and lymphangiogenesis (role in metastasis). Most endothelial markers stain lymphatics and blood vessels without discrimination. Recent marker D2-40 is specific for lymphatic endothelium. This study aims to interpret the utility of D2-40 in detecting LVI, which would be missed on HE and in analysing lymphatic vessel density and LVI as predictive markers for the risk of nodal metastases in colorectal carcinomas. Methods: Study was conducted on 40 specimens of colorectal carcinomas. Immunohistochemistry was performed with D2-40. Stastical analysis was by Spearman’s correlation and Chi square test Result: Mean age was 55.50 years. Of 40 cases, majority were in ascending colon followed by rectum, sigmoid, transverse and descending colon. 80.0% were adenocarcinomas, 15.0% mucinous adenocarcinomas and 5.0% signet-ring cell carcinomas. Tumors were moderately differentiated in 80.0% and poorly in 20.0%. Most patients were in stage T3 followed by T2 and T4. Most common nodal stage was N0 followed by N1, N2 and N3. LVI was detected in eleven more cases on D2-40 than H&E and correlated with lymphatic density and nodal status. Lymphatic density correlated with nodal status and pT. Peritumoral lymphatics were found in 95% cases and intratumoral lymphatics in 90%. Intratumoral lymphatics correlated with nodal status. Conclusion: D2-40 increased the detection rate of LVI as compared to H&E and showed the value of lymphangiogenesis in disease progression and metastasis. DOI: 10.21276/APALM.1122

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

The potential role of perivascular lymphatic vessels in preservation of kidney allograft function

Lymphangiogenesis occurs in diseased native kidneys and kidney allografts, and correlates with histological injury; however, the clinical significance of lymphatic vessels in kidney allografts is unclear. This study retrospectively reviewed 63 kidney transplant patients who underwent protocol biopsies. Lymphatic vessels were identified by immunohistochemical staining for podoplanin, and were classified according to their location as perivascular or interstitial lymphatic vessels. The associations between perivascular lymphatic density and kidney allograft function and pathological findings were analyzed. There were no significant differences in perivascular lymphatic densities in kidney allograft biopsy specimens obtained at 0h, 3months and 12months. The groups with higher perivascular lymphatic density showed a lower proportion of progression of interstitial fibrosis/tubular atrophy grade from 3 to 12months (P for trend=0.039). Perivascular lymphatic density was significantly associated with annual decline of estimated glomerular filtration rate after 12months (r=-0.31, P=0.017), even after adjusting for multiple confounders (standardized β=-0.30, P=0.019). High perivascular lymphatic density is associated with favourable kidney allograft function. The perivascular lymphatic network may be involved in inhibition of allograft fibrosis and stabilization of graft function.

Immunoexpression of VEGFR-3, but not the immunoexpression of VEGF-C or lymphatic density, is correlated with metastasis in lower lip squamous cell carcinoma

The purpose of this study was to evaluate the immunoexpression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3) and their correlation with intratumoural lymphatic density (ILD) and peritumoural lymphatic density (PLD) in metastatic and non-metastatic lower lip squamous cell carcinoma (LLSCC). Twenty-five LLSCC with regional nodal metastasis and 25 LLSCC without metastasis were selected. The percentages of VEGF-C and VEGFR-3 staining in each tumour core and at the deep invasive front were assessed. PLD and ILD were determined using anti-podoplanin antibody. Immunohistochemical findings were correlated with nodal metastasis, clinical staging, local recurrence, clinical outcome, and histological grade. Cytoplasmic immunoexpression of VEGFR-3 in the tumour core was associated with metastasis (P=0.009), patient death (P=0.008), and histological grade (P<0.005). PLD, ILD, and VEGF-C expression showed no significant associations with clinicopathological parameters (P>0.05). PLD and ILD were not significantly correlated with the immunoexpression of VEGF-C or VEGFR-3 (P>0.05). There was a significant positive correlation between PLD and ILD (P=0.004), and between cytoplasmic immunoreactivity of VEGF-C and VEGFR-3 (P=0.011). These results suggest an important role for VEGFR-3 in the progression of LLSCC, and highlight the possible influence of its expression on the prognosis of these tumours. ILD and PLD may not be associated with lymph node metastasis in LLSCC.

Evaluation of hypoxia, angiogenesis, and lymphangiogenesis in actinic cheilitis.

Actinic cheilitis is a potentially malignant condition caused mainly by chronic sun exposure. Here we aim to evaluate the role of hypoxia, angiogenesis, and lymphatic density in the clinical and morphological progression of a series of cases of actinic cheilitis. Immunohistochemistry was used to evaluate positivity to hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-C, and D2-40 in 40 cases of actinic cheilitis of the lower lip. The cases studied exhibited variable degrees of positivity to the markers. The median number of lymphatic vessels was 3.2, 2.4, and 3.0 in lesions showing no epithelial dysplasia (NED) and with mild (MED) and moderate (MOED) epithelial dysplasia, respectively. The median VEGF-C positivity index was 82.44% (NED), 92.74% (MED), and 82.83% (MOED), and the median HIF-1α positivity index was 11.57% (NED), 5.26% (MED), and 13.55% (MOED). No significant differences in lymphatic density or median VEGF-C and HIF-1α positivity indices were observed between histological grades or clinical presentations of actinic cheilitis (P > 0.05). Although representing early events in lip carcinogenesis, the present results suggest that hypoxia, angiogenesis, and lymphangiogenesis do not influence the morphological or clinical progression of actinic cheilitis.

The lymphatic vascular system of the mouse head.

Histological studies of the lymphatic vascular system in adult mice are hampered because bones cannot be sectioned properly. Here, we decalcified the heads of 14-day-old mice, embedded them in paraffin and stained resultant serial sections with the lymphendothelial-specific antibodies Lyve-1 and Podoplanin. We show that the tissues with the highest lymphatic vascular density are the dermis and the oral mucous membranes. In contrast, the nasal mucous membrane is devoid of lymphatics, except for its most basal parts below the vomeronasal organ. The inferior nasal turbinate contains numerous lymphatics and is connected to the nasolacrimal duct (NLD), which is ensheathed by a dense network of lymphatics. The lymphatics of the eye lids and conjunctiva are connected to those of the inferior nasal turbinate. We suggest that cerebro-spinal fluid (CSF) can drain via the optic nerve and NLD lymphatics, whereas CSF drained via the Fila olfactoria into the nasal mucous membrane is used for moisturization of the respiratory air. Tongue, palatine and buccal mucous membranes possess numerous lymphatics, whereas the dental pulp has none. Lymphatics are present in the maxillary gland and close to the temporomandibular joint, suggesting the augmentation of lymph flow by chewing and yawning. Lymphatics can also be found in the dura mater and in the dural septae entering into deeper parts of the brain. Our findings are discussed with regard to CSF drainage and potential routes for ocular tumor dissemination.

Abstract A48: Interactions between lymphatic vessels and cancer-associated fibroblasts in tumor immunity

Abstract Aggressive tumors often hijack the lymphatic vasculature in order to invade and metastasize to their draining lymph nodes (LNs) and ultimately to distant organs. The clinical relevance of lymphatic metastasis has been demonstrated for many cancers, most notably breast cancer, which has been observed to spread preferentially via the lymphogenous route, with the initial sites of metastasis frequently being the tumor-draining LN. Indeed, expression of vascular endothelial growth factor (VEGF)-C, the principal lymphangiogenic growth factor, by the tumor or recruited ancillary cells has been shown to correlate both with LN and distant metastasis, and shorter overall survival, and LN status represents an important prognostic factor for patients diagnosed with breast cancer. Although traditionally considered as passive conduits for tumor cell escape, it is becoming increasingly clear that lymphatic vessels play more active roles in facilitating tumor progression and metastasis. In addition to directly promoting tolerance upon VEGF-C-induced remodeling of the tumor-associated lymphatic vasculature, increased lymphatic density also leads to increased lymph flow from the tumor to the draining LN, causing mechanical stress-induced changes in the stroma surrounding the tumor that may synergize to further suppress anti-tumor immunity. We previously reported that heightened interstitial flow can drive myofibroblast differentiation, commonly identified by the expression of α-smooth muscle actin (α-SMA), and that such flow drives them to align and contract the extracellular matrix in a TGF-β1-dependent manner, leading to stromal stiffening, another feature that is strongly correlated with tumor progression, invasion, and poor prognosis in breast cancer. Here we show that lymphatic endothelial cells (LECs) may contribute directly to stromal stiffening through production of TGF-β1 upon stimulation with VEGF-C. To explore this hypothesis, we developed an orthotopic model of cells isolated from the spontaneous MMTV-PyMT model of breast cancer and stably transfected these cells to over-express VEGF-C, and found that increased VEGF-C secretion correlated with lymphangiogenesis as well as an increase in overall TGF-β1 within these tumors. Furthermore, VEGF-C induces TGF-β1 secretion by LECs in vitro, and in the spontaneous MMTV-PyMT model, inhibition of VEGF-C signaling upon administration of a VEGFR-3 blocking antibody yielded a reduction in overall TGF-β1 in the tumor, as well as specifically in the number of LECs producing TGF-β1 in vivo. Taken together, these results suggest a direct involvement of tumor-associated lymphatic vessels in stromal stiffening during tumor progression, linking tumor mechanobiology with tumor immunology. Citation Format: Ingrid M. van Mier, Jennifer M. Munson, Maria A.S. Broggi, Michele De Palma, Melody A.S. Swartz. Interactions between lymphatic vessels and cancer-associated fibroblasts in tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A48.

Correlation between blood and lymphatic microvascular density and cell proliferation in mouth floor and tongue squamous cell carcinoma

ObjectiveDespite the advances in medicine, the oral squamous cell carcinomas still have high incidence and are very important to study topics such as their lymphatic and microvessel density. This research assessed the correlation between histological grading, formation of new lymphatic and blood vessels and cell proliferation in squamous cell carcinomas (SCC). MethodsTwenty-nine oral floor SCC and 30 tongue cases were assessed for their clinical characteristics and histological grading of malignancy. ResultsThe presence of VEGF (vascular endothelial growth factor VG1), VEGF-C (vascular endothelial growth factor-C), CD105, to determine blood microvascular (MVD), and D2-40 to determine lymphatic density (LMD) was investigated using immunohistochemistry. Histological grading revealed that 73.3% of tongue and 96.67% of floor cases were classified as highly aggressive. Cell proliferation was greater on the floor; however, no significant difference was observed. Most carcinomas were VEGF negative (tongue 63.3% and floor 70.0%) and VEGF-C positive (tongue 73.3% and floor 79.3%). LMD was considerably greater on the tongue. High MVD values occurred in cases with greater cell proliferation. No relationship was determined between the growth factors VEGF and VEGF-C and MVD and LMD, respectively. ConclusionFloor of mouth SCC cases was morphologically more aggressive than tongue cases; however, in tongue carcinomas a greater quantity of lymphatic vessels could represent potential ways for locoregional cell dissemination of the neoplastic cells, independent of histological grading. Blood vascularization presented correlation with cell proliferation in intraoral squamous cell carcinoma and could be useful in the prognostic assessment of this neoplasm.

Platelet interaction with lymphatics aggravates intestinal inflammation by suppressing lymphangiogenesis.

Lymphatic failure is a histopathological feature of inflammatory bowel disease (IBD). Recent studies show that interaction between platelets and podoplanin on lymphatic endothelial cells (LECs) suppresses lymphangiogenesis. We aimed to investigate the role of platelets in the inflammatory process of colitis, which is likely to be through modulation of lymphangiogenesis. Lymphangiogenesis in colonic mucosal specimens from patients with IBD was investigated by studying mRNA expression of lymphangiogenic factors and histologically by examining lymphatic vessel (LV) densities. Involvement of lymphangiogenesis in intestinal inflammation was studied by administering VEGF-receptor 3 (VEGF-R3) inhibitors to the mouse model of colitis using dextran sulfate sodium and evaluating platelet migration to LVs. The inhibitory effect of platelets on lymphangiogenesis was investigated in vivo by administering antiplatelet antibody to the colitis mouse model and in vitro by coculturing platelets with lymphatic endothelial cells. Although mRNA expressions of lymphangiogenic factors such as VEGF-R3 and podoplanin were significantly increased in the inflamed mucosa of patients with IBD compared with those with quiescent mucosa, there was no difference in LV density between them. In the colitis model, VEGF-R3 inhibition resulted in aggravated colitis, decreased lymphatic density, and increased platelet migration to LVs. Administration of an antiplatelet antibody increased LV densities and significantly ameliorated colitis. Coculture with platelets inhibited proliferation of LECs in vitro. Our data suggest that despite elevated lymphangiogenic factors during colonic inflammation, platelet migration to LVs resulted in suppressed lymphangiogenesis, leading to aggravation of colitis by blocking the clearance of inflammatory cells. Modulating the interaction between platelets and LVs could be a new therapeutic means for treating IBD.

Lymphangiogenesis is increased in heart valve endocarditis

BackgroundInflammation-associated lymphangiogenesis (IAL) has been identified as part of several acute and chronic inflammation. Sparse data exist on lymphatics during endocarditis. Materials and methodsFifty-two patients with surgically resected valves were included. Endocarditis was present in 18 aortic and 10 mitral valves. Controls consisted of 15 degenerative aortic and 9 degenerative mitral valves. There were 22 males with endocarditis and 17 males in controls. The mean age was 58 (SD 15) years with endocarditis vs. 62 (SD 13) years for controls. Lymphatics were detected by podoplanin antibody immunohistochemistry and morphometrical analysis was performed. ResultsThe lymphatic density in endocarditis was 833 (SD 529) vessels/mm2 (range 0–1707) as compared with 39 (SD 60) vessels/mm2 (range 0–250) in controls (p=0.000). In endocarditis, the mean lymphatic size was 153 (SD 372) μm2 ranging from 1 to 2034μm2, whereas it was 30 (SD 29) μm2, with maximum 90μm2 and minimum 2μm2 in controls (p=0.000). ConclusionsIAL is increased in valves with endocarditis as compared with controls. Lymphatics in heart valves may provide a novel means for treatment strategies against endocarditis.

Increased Angiogenesis and Lymphangiogenesis in Metastatic Sentinel Lymph Nodes Is Associated With Nonsentinel Lymph Node Involvement and Distant Metastasis in Patients With Melanoma.

Lymph node angio- and lymphangio-genesis have been shown to play an important role in the premetastatic niche of sentinel lymph nodes. In the current study we have investigated the association of angio- and lympangio-genesis related parameters in metastatic sentinel lymph nodes of patients with melanoma with the presence of nonsentinel and distant organ metastasis. Peritumoral and intratumoral relative blood and lymphatic vessel areas (evaluated by Chalkley method), blood and lymphatic microvessel densities, and the rates of blood and lymphatic vessel proliferation were assessed in primary tumors and sentinel lymph node metastasis of 44 patients with melanoma using CD34/Ki-67 and D240/Ki-67 immunohistochemical double staining. Primary melanoma exhibited significantly higher rate of lymphatic proliferation compared with its lymph node metastasis (P < 0.05), while lymph node metastasis showed significantly higher rate of blood vessel proliferation (P < 0.05). Using multivariate logistic regression model, the rate of peritumoral lymphatic proliferation was inversely associated with positive nonsentinel lymph node status (P < 0.05), whereas the rate of intratumoral blood vessel proliferation was associated with distant organ metastasis (P < 0.05). Using multivariate Cox regression analysis, the rate of intratumoral blood vessel proliferation was also inversely associated with overall survival of patients with melanoma (P < 0.05).

Patient-derived xenograft models of squamous cell carcinoma of the uterine cervix

Patient-derived xenograft (PDX) models of cancer are considered to reflect the biology and treatment response of human tumors to a larger extent than xenograft models initiated from established cell lines. The characterization of a panel of four novel PDX models of cervical carcinoma of the uterine cervix is described in this communication. The outcome of treatment differed substantially among the donor patients, and the PDX models were found to mirror the histology, aggressiveness, and metastatic propensity of the donor patients' tumors. Two of the models (BK-12 and LA-19) were highly metastatic, one model (ED-15) was poorly metastatic, and one model (HL-16) was non-metastatic. The primary tumors of the two highly metastatic models showed high density of intratumoral lymphatics, whereas the other two models did not develop intratumoral lymphatics. The potential of the models to metastasize to lymph nodes was associated with high expression of both angiogenesis-related genes and cancer stem cell-related genes. The models may be highly valuable for studying mechanisms linking lymph node metastasis to lymphangiogenesis, hemangiogenesis, and the presence of cancer stem cells.

IGFBP7 functions as a potential lymphangiogenesis inducer in non-small cell lung carcinoma.

Lymphangiogenesis is not only involved in the processes of embryonic development, tissue repair and chronic inflammation, but also in tumor lymphatic metastasis. Metastatic tumor cells spreading through lymphatic vessels occur in non-small cell lung carcinoma (NSCLC), with regional lymph node metastasis often being the most important prognostic factor for carcinoma patients. Recent research has identified a range of lymphangiogenic growth factors that could conceivably play a great role in promoting tumor lymphangiogenesis and lymphatic metastasis. The most extensively accepted signaling pathways promoting lymphangiogenesis in tumors include the secreted lymphangiogenic proteins: vascular endothelial growth factor-C (VEGF-C) and VEGF-D, and their cognate receptor on lymphatic endothelium VEGF receptor-3 (VEGFR-3). Targeting VEGF pathway strategy sometimes failed to decrease tumor metastasis in vivo experiments and clinical trials. It is unclear whether the tumor cells induced the lymphangiogenesis process, while VEGF pathway could not completely illustrate the mechanism of tumor cell lymphatic metastasis. To explore the novel tumor lymphangiogenesis targets, we screened 181 candidate genes between high lymphatic vascular density (LVD) and low LVD in lung adenocarcinomas using Human Genome U133 Plus 2.0 Microarray. Insulin-like growth factor binding protein 7 (IGFBP7) was proven to be associated with metastatic clinicopathological features and high LVD. Furthermore, by assessing the capability of lymphatic endothelial cell forming lymphatic vessel-like structures in vitro, it appears to enhance lymphangiogenesis.

Abstract 12624: Therapeutic Lymphangiogenesis Improves Diastolic Function by Limiting Cardiac Edema and Fibrosis After Myocardial Infarction

Little knowledge exists on how cardiac lymphatics respond to myocardial infarction (MI). We investigated the impact of MI on cardiac lymphatic structure and function during the development of chronic heart failure (CHF). We hypothesized that 1) insufficient cardiac lymph drainage may contribute to the deleterious cardiac remodeling post-MI; and 2) therapeutic stimulation of lymphatic growth, or lymphangiogenesis, may reduce chronic myocardial edema and inflammation in this setting. MI was modeled in Wistar rats by coronary occlusion (45 min) followed by reperfusion. Lymphangiogenesis-selective VEGF-C was delivered intramyocardially using slow-release albumin-alginate microparticles. Animals were divided into: sham (n=19), control MI (n=64), and treated MI, receiving low (1.5 μg; n= 54) or high (5 μg; n=26) dose of VEGF-C upon reperfusion. Cardiac function, lymphatics, edema, and levels of infiltrating immune cells were evaluated at 3 and 8 weeks post-MI. We found that control rats displayed lymphatic remodeling with increased lymphatic capillary density but rarefaction of epicardial pre-collector and collector vessels, leading to cardiac lymphatic dysfunction. In accordance with insufficient lymph drainage, chronic myocardial edema and low grade inflammation were found at both 3 and 8 weeks post-MI as determined by gravimetry, MRI T2 imaging, and immunohistochemistry, respectively. VEGF-C treatment led to a dose-dependent increase in lymphatic capillary density (sham, 255±16; con, 267±25; VEGF-C, 362±22 lymphatics/mm 2 ; p&lt;0.05) and reduced rarefaction of pre-collectors and collector vessels. As a consequence, myocardial edema (sham, 75.5±0.1; con, 77.9±0.2; VEGF-C, 77.3±0.1% water content; p&lt;0.05) and macrophage infiltration levels were both significantly reduced. Further, cardiac interstitial fibrosis was completely prevented by 8 weeks, even by low dose VEGF-C (con, 6.0±0.6; VEGF-C, 3.3±0.1% fibrosis; p&lt;0.001), leading to reduced diastolic dysfunction (LVDPVR, LV end-diastolic pressure-volume relation: sham, 0.8±0.1; con, 2.0±0.2; VEGF-C, 1.1±0.1; p&lt;0.001). In conclusion, our data suggest that therapeutic lymphangiogenesis may represent a new therapeutic target in CHF.