miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity.

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor ß1 (TGF-ß1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-ß1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-ß1-induced α-SMA expression. These data demonstrate that TGF-ß1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-ß1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.

Comparison of avascular lymph node fragment transplantation techniques to optimize lymphangiogenesis in the minipig model.

BACKGROUND: Secondary lymphoedema is a challenging pandemic. This condition may arise after oncologic resection of tumor-draining lymph nodes and/or radiation. Plastic-surgical procedures for lymphoedema comprise transplantation of vascularized lymph node flaps, which are, however, technically challenging and difficult to implement on a global level due to the scarcity of microsurgery facilities in some countries. To improve this situation, comparative research in valid animal models is needed. METHODS: A total of 33 minipigs were subjected to lymphatic resection in the hind limbs. This large animal model was used in a first phase to compare different lymph node fragmentation methods and assess lymphatic regeneration after avascular transplantation. In a second phase, several stimulants were tested for their effect on lymphatic regeneration after fragment transplantation. In a third phase, animals additionally received irradiation of the groin. In this novel animal model, autologous avascular lymph node fragment transplantation was complemented by peripheral injections of vascular endothelial growth factor-C (VEGF-C). Finally, regeneration rates were quantified in relative numbers (percentage) in the irradiated tissue. RESULTS: In the first phase, transversal lymph node fragmentation under preservation of the nodal capsule showed the best percentage of regeneration (62.5%). Peripheral intradermal administration of VEGF-C enhanced lymph node fragment regeneration (70.8%) better than injections of tetanus toxoid (41.6%) or Streptococcus suis (62.5%). Lymph node fragment regeneration also occurred in an irradiated porcine model of lymphadenectomy under VEGF-C administration (66.6%). CONCLUSIONS: The present findings provide a pre-clinical proof-of-concept for a possible simplification strategy for current operative procedures of autologous lymph node transplantation.Level of evidence : Not gradable.

Age-dependent histoarchitectural changes in human lymph nodes: an underestimated process with clinical relevance?

Experimental evidence indicates that lymph nodes in humans undergo alterations during ageing. This is clinically important because of the crucial role of these organs in the immune system and their lymph reabsorption and drainage function. Although some age-related changes in lymph node histoarchitecture have been described, they are seldom taken into account in traditional depictions of lymph nodes. Recently introduced clinical procedures, such as intranodal vaccination or lymph node transplantation, have demonstrated the need for an accurate knowledge of these degenerative processes. In this study, superficial inguinal lymph nodes were obtained from 41 deceased patients between 17 and 98 years old. To minimize immunological influences, such as chronic diseases, specimens were only obtained from forensic pathology autopsies. An immunohistochemical analysis was carried out, on the basis of which lymph node degeneration was scored according to the numbers of lymphocytes and high endothelial venules, and degree of fibrosis and lipomatosis. We observed an age-dependent tendency towards the replacement of areas populated with diverse immune cells by connective tissue. Paradoxically, these changes were also detected in some of the nodes from younger age groups. In conclusion, lymph nodes can display degenerative changes that are mainly age-related and often diverge from the common description found in textbooks. These alterations should be taken into account when dealing with lymph nodes diagnostically and therapeutically in clinical practice.

Comprehensive genetic and functional characterization of IPH-926: a novel CDH1-null tumour cell line from human lobular breast cancer.

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E-cadherin. ILBCs also lack beta-catenin expression and show aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin. The lack of a well-characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH-926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH-926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH-926 expressed various epithelial cell markers but lacked expression of E-cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH-926 from this particular tumour. IPH-926 also lacked beta-catenin expression and showed aberrant cytoplasmic localization of p120-catenin. Array-CGH analysis of IPH-926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH-926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH-926 cells were anti-cancer drug-resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH-926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH-926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo.

Regeneration of autotransplanted avascular lymph nodes in the rat is improved by platelet-rich plasma.

The aim of this study was to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, in analogy to results obtained in other species. This rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. The results show for the first time that the rat is an adequate model to study the regeneration of transplanted lymph nodes. Lymph node fragmentation seems to affect transplant regeneration negatively. An immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. However, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. Lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. Acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. This disease causes lifelong disability due to chronic swelling and increased risk of infections. It currently lacks an effective treatment.

Frequency and risk factors for arm lymphedema after multimodal breast-conserving treatment of nodal positive breast Cancer - a long-term observation.

BACKGROUND: Arm-lymphedema is a major complication after breast cancer. Recent studies demonstrate the validity of predicting Breast Cancer Related Lymphedema (BCRL) by self-reports. We aimed to investigate the rate of BCRL and its risk factors in the long-term using self-reported symptoms. METHODS: Data was collected from 385 patients who underwent multimodal therapy for nodal positive breast cancer, including breast conserving surgery, axillary dissection, and local or locoregional radiotherapy. Two validated questionnaires were used for the survey of BCRL (i.e. LBCQ-D and SDBC-D). These were analysed collectively with retrospective data of our medical records. RESULTS: 23.5% (n = 43) suffered a permanent BCRL (stage II-III) after a median follow-up time of 10.1 years (4.9-15.9 years); further 11.5% (n = 23) reported at least one episode of reversible BCRL (Stage 0-I) during the follow-up time. 87.1% of the patients with lymphedema developed this condition in the first two years. Adjuvant chemotherapy was a significant risk factor for the appearance of BCRL (p = 0.001; 95%-CI 7.7-10.2). CONCLUSIONS: Breast cancer survivors face a high risk of BCRL, particularly if axillary dissection was carried out. Almost 90% of BCRL occurred during the first two years after radiotherapy. Self-report of symptoms seems to be a suitable instrument of early detection of BCRL.

Worldwide assessment of healthcare personnel dealing with lymphoedema.

BACKGROUND: Lymphoedema is a pandemic with about 250 million people suffering from this condition worldwide. Lymphatic diseases have considerable public health significance, but yet few professionals are specialised in their management causing a substantial burden on health resources. AIMS AND OBJECTIVES: This study aims to give an overview of the approximate number of medical professionals, professional societies, institutions and companies dealing with lymphoedema in various countries. Concepts of improvement for current human resources are considered. METHODS: An online database analysis (Google search engine and PubMed) was carried out for each country of the world. Additionally, relevant congress participant lists as well as member lists of significant medical societies and reports of the World Health Organisation were analysed. RESULTS: Overall distribution of tertiary level professionals specialised in this field is heterogenous. A decrescent gradient of professionals can be seen between developed and developing countries and between urban and rural areas. Countries in general do not seem to have yet met the current demand for specialists at tertiary level in this field. CONCLUSIONS: This study intends to draw attention to the current medical coverage gaps due to a low number of lymphoedema specialists at tertiary level. It wishes to start a discussion about structured reimbursement and certification of knowledge and skills that are essential incentives for experts to act as multiplicators and change the lack of care in the mid-term. Current fail prescriptions and evitable disability and sick certificates represent a high financial burden that could be reinvested in a correct management. Policy makers must focus in the two above mentioned essential measures. Medical training and the consequent development of the industry will then naturally take place, as it was the case for other professional groups in the past.

Effect of cryopreservation on lymph node fragment regeneration after autologous transplantation in the minipig model.

INTRODUCTION: Lymphoedema is a worldwide pandemic causing swelling of tissues due to dysfunctional transport of lymph fluid. Present management concepts are based in conservative palliation of symptoms through manual lymphatic drainage, use of compression garments, manual lymph drainage, exercise, and skin care. Nevertheless, some curative options as autologous lymph node transplantation were shown to reduce lymphoedema in selected cases. Lately, some concern has arisen due to reports of donor site morbidity. A possible solution could be the development of artificial lymph node scaffolds as niches of lymphatic regeneration. Engineering these scaffolds has included cryopreservation of lymph node stroma. However, the effects of cryopreservation on the regeneration capacities of these organs were unknown. MATERIALS AND METHODS: Here, we used the minipig animal model to assess lymphatic regeneration processes after cryopreservation of autologous lymph nodes. Superficial inguinal lymph nodes were excised and conserved at -80°C for 1 month. Thereafter, lymph node fragments were transplanted in the subcutaneous tissue. RESULTS: Regeneration of the lymph nodes was assessed five months after transplantation. We show that lymph node fragment regeneration takes place in spite of former cryopreservation. Transplanted fragments presented typical histological appearance. Their draining capacity was documented by macroscopic transport of Berlin Blue dye as well as through SPECT-CT hybrid imaging. DISCUSSION: In conclusion, our results suggest that processes of cryopreservation can be used in the creation of artificial lymph node scaffolds without major impairment of lymph node fragments regeneration.

Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema.

Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

VEGF-C improves regeneration and lymphatic reconnection of transplanted autologous lymph node fragments: An animal model for secondary lymphedema treatment.

Secondary lymphedema occurs after for example breast cancer surgery and radiation in 20-50% of the patients. Due to the poor outcomes of surgical treatments in the past, the therapy often remains symptomatic. However, avascular transplantation of autologous lymph node fragments (LN-Tx) combined with postoperative injections of vascular endothelial growth factor-C (VEGF-C) emerges as a potential surgical therapy. In this study, adult rats underwent LN-Tx to investigate the following parameters of VEGF-C application: time point, location and dosage. Furthermore, the influences of VEGF-C on lymphatic reconnection and transplant regeneration were analyzed. The reconnection was investigated using intradermally injected blue dye and the regeneration was evaluated histologically using hematoxylin-eosin (H&E) staining and immunohistochemistry. The higher dosage enhanced the reconnection rates significantly and showed a statistical tendency of improving regeneration. An application on early postoperative days and the injection into the medial thigh improved the reconnection significantly. However, these variables did not affect the regeneration statistically. This study confirms that LN-Tx combined with lymphatic growth factor VEGF-C is a possible approach in the therapy of secondary lymphedema and shows the important role of VEGF-C application parameters.

Surgical procedures in lymphedema management.

OBJECTIVE: Lymphedema has a high incidence and various causes. It reduces patients' quality of life and productivity and currently lacks a cure. Management is based on lifelong physical therapies. Many surgical procedures have been proposed for lymphedema without significant acceptance. This study evaluates surgical procedures aimed at the management of lymphedema and highlights present evidence. METHODS: Based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement and the Grading of Recommendations Assessment, Development and Evaluation consensus, a systematic literature search (MEDLINE and The Cochrane Library) was performed to identify studies evaluating treatment outcomes after lymphedema surgery. The authors evaluated all articles found with the keywords "lymphedema" and "surgery," including experimental studies in animals. Cross referencing was made. Next, a protocol was created to assess the degree of quality of publications in this field. An overview of the complete medical literature was performed. Thereafter, publications meeting inclusion criteria were attributed a score according to the assessment protocol. This allowed an overview of the scientific quality of all surgical procedures for lymphedema. RESULTS: A total of 108 article texts were read and 70 publications included in this study according to predefined criteria. Lymphedema operative procedures were classified according to type. The outcomes highlighted the importance of individual patient analysis, as most interventions are not sufficiently studied to sustain clinical recommendations. CONCLUSIONS: Risk factors for acquired lymphedema, such as lymph node excision and radiation therapy, are well identified and should allow primary prevention. Improved diagnosis, classification, standardized volume measurement, staging, and follow-up of lymphedema patients can facilitate their management and allow valid retrospective studies. Currently, there is no evidence of any treatment yielding high long-term cure rates. Therefore, lymphedema management must be based on interdisciplinary approaches, with curative or palliative therapy options discussed openly with the patient. Therapeutic plans should not exclude surgery. Nevertheless, additional studies are recommended to prove the validity of some surgical approaches.

Quantification of lymphedema in a rat model by 3D-active contour segmentation by magnetic resonance imaging.

Secondary lymphedema is a common complication after lymph node excision and radiotherapy in cancer therapy. Therapies are limited to symptomatic treatment. Adequate animal models to test potential surgical therapies are needed. The aim of this study was to induce a tissue environment in the hind leg of the rat similar to the one found in operated and irradiated patients. Quantification of edematous swelling was performed by an automatic 3D-contour segmentation (ITK- Snap ©) on MR- images. Swelling was induced by excision of superficial inguinal and popliteal lymph nodes and adjacent lymphatic vessels, followed by radiotherapy of the right groin with a single dose of 15 Gy. Four weeks after irradiation, the animals were examined with MRI of both hind legs. Fluid volumes around the joint line of the knee were calculated on T2-weighted images. We documented a significant higher volume of fluid in the legs following excision of lymph nodes and lymphatic vessels, combined with radiotherapy than in control legs.

Improved regeneration of autologous transplanted lymph node fragments by VEGF-C treatment.

Secondary lymphedema is a common complication after removal of lymph nodes in combination with radiation therapy in the treatment of breast cancer, cervical cancer, and melanomas. Only symptomatic therapies are available at the moment, and lymphedema is for most patients a lifelong condition involving psychological and physical disabilities. Animal models exist to study the pathophysiology of lymphedema but not to study surgical treatments. The aim of this study was to show that regeneration of autologous transplanted lymph node fragments is possible in rats that were irradiated previously locally in the groin and to examine the effects of vascular endothelial growth factor (VEGF)-C injections on the rate of regeneration of transplanted lymph nodes. In all of the animals, inguinal and popliteal lymph nodes and adjacent lymphatic vessels were unilaterally removed and the inguinal region irradiated by a single dose of 15 Gy. Afterward, lymph node fragments were transplanted subcutaneously in the irradiated region. Half of the animals were treated by local VEGF-C injections after transplantation. Four weeks after transplantation, drainage of the leg was tested by injection of blue dye, and the transplanted fragments were removed and examined immunohistologically. We could show that regeneration of autologous transplanted lymph node fragments is possible in areas treated with radiotherapy in the rat. We also documented that transplants can achieve a connection to the lymphatic collectors of the leg. The results suggest that the outcome of regeneration can be improved by injection of VEGF-C in the transplantation area. Thus, lymph node fragment regeneration may be relevant for lymphedema prevention and therapy.

Acquired lymphedema: an urgent need for adequate animal models.

In cancer patients, the removal of tumor-draining lymph nodes during tumor resection can lead to acquired lymphedema. This disease, which is characterized by tissue swelling and increased risk of infection due to restricted lymph flow, lacks an effective treatment. Limitations to the design and conduct of randomized trials to date have limited the evaluation of proposed surgical techniques. As a result, animal models have provided an important research base. This review summarizes work in canine, rabbit, and rodent models of acquired lymphedema, focusing on arising limitations and potential applications.

Donor-site morbidity of ear cartilage autografts.

BACKGROUND: The external ear provides a versatile cartilage source for reconstructive procedures, especially for augmentative rhinoplasty. The authors evaluated the short- and long-term morbidity associated with ear cartilage harvest using concha, tragus, and scapha as donor sites. METHODS: The study included 52 patients in whom a cartilage graft from the external ear was harvested between February of 2001 and June of 2005. Donor-site morbidity was assessed in the early postoperative follow-up period. Twenty-eight patients were examined within a follow-up time of 3 to 168 months. Long-term morbidity assessment included documentation of patients' subjective complaints, clinical examination of the donor site, and anthropometric measurements. RESULTS: The relevant morbidity factors in the early postoperative period were hematoma formation (6.7 percent) and sensory impairment (3.3 percent). In long-term follow-up, sensory impairment was the most frequent condition objectively assessed and subjectively complained of (12.9 percent). Overall, sensory impairment was confined to concha as the donor site. Anthropometric measurements showed a mean difference in the length of the affected ear compared with the contralateral ear of 1.8 mm, a width difference of 2.5 mm, a difference in tragus/lateral canthus distance of 1.4 mm, and a difference in protrusion angle of 2.4 degrees. Statistically significant differences between values obtained from operated and nonoperated ears could not be detected for every evaluated parameter using the paired t test. Aesthetically relevant complications were rare and their occurrence restricted to single cases. CONCLUSION: Cartilage graft harvest from the auricle can be considered as a relatively safe procedure with a favorable aesthetic outcome.