The lymphatic vasculature revisited

Lymphatic vessels constitute a ubiquitous countercurrent system to the blood vasculature that returns interstitial fluid, salts, small molecules, resorbed fat, and cells to the bloodstream. They serve as conduits to lymph nodes and are essential for multiple physiologic activities. However, they are also hijacked by cancer cells to establish initial lymph node metastases, as well as by infectious agents and parasites. Despite these obvious important functions in human pathologies, a more detailed understanding of the molecular mechanisms involved in the regulation of the lymphatic vasculature has trailed that of the blood vasculature for many years, mainly because critical specific characteristics of lymphatic endothelial cells were discovered only recently. In this Review series, several major aspects of the active and passive involvement of the lymphatic vasculature in human disease and physiology are presented, with a focus on translational findings.Many scientific fields develop in waves that vary in frequency and amplitude, with rapid progress initiated by serendipitous findings in seemingly unrelated fields. Research on the structure and function of the lymphatic vasculature exemplifies this model. As presented in this Review series, novel insights into the involvement of lymphatic vessels in human disease and physiology have not only provided additional information on pathogenesis, but have changed the prevailing paradigms of disease mechanisms as a prelude to improved targeted therapies.Historically, the identification of lymphatic vessels relied on their content, known as “white blood” (1). Their macroscopic visualization was made possible by intradermal injection of colloid tracers in fresh cadavers, and striking examples of the results are provided by the anatomical wax models produced in Florence around 1770 and on display in the Josephinum in Vienna ( www.josephinum.ac.at). A model of the cervical lymphatic network is depicted on the cover of this series. These techniques primarily detect large branches of the lymphatic system, i.e., the collecting vessels that serve as peristaltic pumps to move the lymphatic fluid toward the thoracic duct by a precisely coordinated contraction of the smooth muscles in their media, while strategically positioned valves prevent reflux. Recent advances in our understanding of smooth muscle contraction (2) and formation and stability of the valves (3) have clarified these processes. In this series, Eva Sevick-Muraca and colleagues (4) summarize the current state of in vivo visualization that reveals the lymphatic vasculature in much better resolution. Though our understanding of the collecting vessels has improved, we are progressively gaining detailed knowledge concerning the initial microvascular segment, which is arguably the most functionally relevant part of the vascular lymphatic tree and hence the topic of most Reviews in this series. Under the microscope the initial lymphatic capillaries are composed of sacs of endothelial cells and are devoid of pericytes. These capillaries merge into a precollector vessel segment with few pericytes that opens into the collectors (Figure ​(Figure11). Open in a separate windowFigure 1Basic design of the initial segment of the lymphatic microvasculature of the dermis.The initial capillaries start as blind sacs. Their LECs form overlapping junctions, express large amounts of the membrane mucoprotein podoplanin (green), and release the chemokine CCL21. This attracts CCR7⁺ immune cells, such as dendritic cells and Tregs. The precollectors contain LECs with low amounts of podoplanin, and the LECs produce CCL27, which attracts inflammatory CCR10⁺ T lymphocytes. This precollector segment opens into the collecting vessels that are endowed with podoplanin-low endothelial cells and that form valves. Pericytes/mural cells partially cover the precollectors and completely ensheath collecting vessels. At the center of the current increase in lymphatic research are new insights into the biology of the lymphatic endothelial cells (LECs). This phase was heralded by detailed ultrastructural investigations of initial lymphatic vessels (5). The critical breakthrough to a functional understanding was achieved with the discovery of the main lymphangiogenic growth factor, VEGFC, and its receptor, VEGFR3 (6). This was followed by the identification of proteins that discriminate between endothelial cells of the lymphatic and blood vessel lineages (7) that can serve as selective markers for the localization and isolation of LECs (8, 9), a development reviewed by Kari Alitalo and colleagues in this series (10). Since the lymphatic system is as branched and widely distributed as the blood vasculature, it is not surprising that it is involved in most disease processes, ranging from inflammation to metastatic spreading of cancers. It is becoming increasingly clear that the lymphatics do not just serve as passive conduits for interstitial fluid and cells, but are actively involved in disease, as reviewed in detail in this series. Below, I highlight a few selected aspects of current research that could become clinically relevant in the coming years.     

Everolimus inhibits glomerular endothelial cell proliferation and VEGF, but not long-term recovery in experimental thrombotic microangiopathy.

BACKGROUND: Everolimus is a potent immunosuppressant used in renal transplant therapy, but its effects on renal endothelial cell regeneration after injury are unknown. The effects of an everolimus therapy were investigated in a model of renal thrombotic microangiopathy (TMA) with specific endothelial cell (EC) injury in the rat in vivo as well as in glomerular ECs in vitro. METHODS: During the early regenerative phase (day 3) of the renal microvascular injury model in vivo, everolimus inhibited glomerular EC proliferation by up to 60% compared with vehicle-treated rats, whereas apoptosis was not different in these groups. This decreased EC proliferation was associated with an enhanced deposition of fibrin in everolimus treated animals on day 3. In cultured glomerular endothelial cells, everolimus effectively and dose dependently inhibited cellular proliferation. This anti-proliferative effect was associated with a reduced phosphorylation of the p70S6 kinase and reduction of the pro-angiogenic factor VEGF in glomeruli in vivo and in cultured podocytes in vitro. RESULTS: Despite the prolonged EC repair and in contrast to the anti-Thy1 nephritis model, everolimus therapy did not disturb the long-term repair reaction in this thrombotic microangiopathy model. CONCLUSION: Everolimus is anti-proliferative for glomerular EC in vitro and in vivo and does not seem to have detrimental long-term effects in experimental renal TMA, when only the glomerular endothelium, but not the mesangium is severely injured.     

Cellular pathology of Heyman nephritis: monoclonal antibodies against the pathogenic antigen Gp 330

Heymann nephritis is an experimental auto-immune glomerulonephritis, closely resembling human epimembranous nephritis, which is induced by identified antigens in the brush border membranes of kidney proximal tubules. The hallmark of the disease is the accumulation of immune deposits in a granular pattern in the lamina rara externa of the glomerular basement membrane. We have established that a large membrane glycoprotein with an apparent molecular weight of 330 kDal (pg 330) is the pathogenic antigen. By means of immunohistochemistry, using mono- and polyclonal anti-pg 330 IgG we found that gp 330 is present in the cell membranes of glomerular epithelial cells and that it is concentrated there in coated pits (specialized areas off the cell membrane which play a key role in receptor mediated endocytosis for ligands such as insulin and others). On the basis of these findings we propose the following mechanism of formation of an immune deposit: (1) "In-situ" formation of immune complexes of gp 330 and anti-gp 330 IgG; (2) shedding of the immune complexes into the basement membrane and (3) crosslinking of the immune complexes into the basement membrane. This scheme could be a general mechanism by which immune deposits are formed also in various other auto-immune diseases.     

The crucial role of macrophages in lymphangiogenesis

Lymphangiogenesis is associated with pathological processes such as the metastatic spread of carcinoma cells and organization of immunologically active lymphocytic infiltrates following organ transplantation. It has not yet been established whether expansion of the lymphatic vascular meshwork is driven by incorporation of progenitor cells or by local endothelial cell division. In this issue of the JCI, Maruyama et al. provide evidence that after mouse corneal transplant, CD11b+ macrophages infiltrate the corneal stroma and transdifferentiate into lymphatic endothelial cell clusters that join existing lymphatic vessels. In complementary in vitro experiments, murine peritoneal macrophages expressed lymphatic endothelial markers and formed vessel-like protrusions. These findings add yet another facet to the plasticity of macrophages, which are already known to transform from naive monocytes into VEGF-C-producing cells. Thus, macrophages support lymphangiogenesis in 2 different ways, either by transdifferentiating and directly incorporating into the endothelial layer or by stimulating division of preexistent local lymphatic endothelial cells.     

Radiogenic lymphangiogenesis in the skin

The time course of microvascular changes in the environment of irradiated tumors was studied in a standardized human protocol. Eighty skin biopsies from 40 patients with previously treated primary breast cancer were taken from irradiated skin and corresponding contralateral unirradiated control areas 2 to 8 weeks, 11 to 14 months, or 17+ months after radiotherapy (skin equivalent dose 30 to 40 Gy). Twenty-two biopsies of 11 melanoma patients who had undergone lymph node dissection were used for unirradiated control. We found an increase of total podoplanin(+) lymphatic microvessel density resulting mainly from a duplication of the density of smallest lymphatic vessels (diameter <10 microm) in the samples taken 1 year after radiation. Our findings implicate radiogenic lymphangiogenesis during the 1st year after therapy. The numbers of CD68(+) and vascular endothelial growth factor-C(+) cells were highly elevated in irradiated skin in the samples taken 2 to 8 weeks after radiotherapy. Thus, our results indicate that vascular endothelial growth factor-C expression by invading macrophages could be a pathogenetic route of induction of radiogenic lymphangiogenesis.     

The human glomerular podocyte is a novel target for insulin action

Microalbuminuria is significant both as the earliest stage of diabetic nephropathy and as an independent cardiovascular risk factor in nondiabetic subjects, in whom it is associated with insulin resistance. The link between disorders of cellular insulin metabolism and albuminuria has been elusive. Here, we report using novel conditionally immortalized human podocytes in vitro and human glomeruli ex vivo that the podocyte, the principal cell responsible for prevention of urinary protein loss, is insulin responsive and able to approximately double its glucose uptake within 15 min of insulin stimulation. Conditionally immortalized human glomerular endothelial cells do not respond to insulin, suggesting that insulin has a specific effect on the podocyte in the glomerular filtration barrier. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1 and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. Insulin responsiveness in this key structural component of the glomerular filtration barrier may have central relevance for understanding of diabetic nephropathy and for the association of albuminuria with states of insulin resistance.     

R-roscovitine (CYC202) alleviates renal cell proliferation in nephritis without aggravating podocyte injury

BACKGROUND: Cyclin-dependent kinase (CDK) inhibition is a new therapeutic approach to proliferative glomerulonephritides. CDK2 is required for G(1)/S transition and DNA synthesis and is inhibited by CYC202 (R-roscovitine). Since podocytes express CDK2 in nephritis and since loss of podocytes contributes to glomerulosclerosis, the rationale of the present study was to test whether CDK2 inhibition is safe in instances of podocyte injury. METHODS: Rats with passive Heymann nephritis, a model of membranous glomerulonephritis, were treated (day 3 to 30) with vehicle, low (25 mg/kg/day), or high (50 mg/kg/day) doses of CYC202. RESULTS: On day 27, blood pressure was normal in nephritic controls and was dose-dependently reduced by CYC202. Urinary albumin excretion did not differ between the groups on days 9, 16, 23, and 30. To investigate podocyte injury, we assessed the glomerular de novo expression of desmin, which was markedly up-regulated in almost all passive Heymann nephritis glomeruli but was not significantly different between the three groups. No tubulointerstitial de novo expression of desmin or alpha-smooth muscle actin (alpha-SMA), or tubulointerstitial monocyte/macrophage infiltration was noted in any group. Biologic activity of CYC202 was evident in the form of a dose-dependent decrease in the number of glomerular and tubulointerstitial mitotic figures as compared to vehicle alone. Glomerular immunostaining for cyclin D1, a marker for G(0) to G(1) transition, was significantly decreased in CYC202 treated groups at day 9. CONCLUSION: Whereas inhibition of CDKs by CYC202 reduced intrarenal cell proliferation in passive Heymann nephritis it did not aggravate podocyte damage, suggesting that this novel therapeutic approach is safe in renal diseases characterized by podocyte injury.     

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.