Increased Interstitial Protein Because of Impaired Lymph Drainage Does Not Induce Fibrosis and Inflammation in Lymphedema.

Jan 2103

Markhus CE, Karlsen TV, Wagner M, Svendsen OS, Tenstad O, Alitalo K, Wiig H.

Source

Department of Biomedicine, University of Bergen, Norway.

Abstract

OBJECTIVE:

The pathophysiology of lymphedema is incompletely understood. We asked how transcapillary fluid balance parameters and lymph flow are affected in a transgenic mouse model of primary lymphedema, which due to an inhibition of VEGFR-3-Ig signaling lacks dermal lymphatics, and whether protein accumulation in the interstitium occurring inlymphedema results in inflammation.

METHODS AND RESULTS:

As estimated using a new optical-imaging technique, we found that this signaling defect resulted in lymph drainage in hind limb skin of K14-VEGFR-3-Ig mice that was 34% of the corresponding value in wild-type. The interstitial fluid pressure and tissue fluid volumes were significantly increased in the areas of visible swelling only, whereas the colloid osmotic pressure in plasma, and thus the colloid osmotic pressure gradient, was reduced compared to wild-type mice. An acute volume load resulted in an exaggerated interstitial fluid pressure response in transgenic mice. There was no accumulation of collagen or lipid in skin, suggesting that chronic edema presented in the K14-VEGFR-3-Ig mouse was not sufficient to induce changes in tissue composition. Proinflammatory cytokines (interleukin-2, interleukin-6, interleukin-12) in subcutaneous interstitial fluid and macrophage infiltration in skin of the paw were lower, whereas the monocyte/macrophage cell fraction in blood and spleen was higher in transgenic compared with wild-type mice.

CONCLUSIONS:

Our data suggest that a high interstitial protein concentration and longstanding edema is not sufficient to induce fibrosis and inflammation characteristic for the human condition and may have implications for our understanding of the pathophysiology of this condition.

A M A

Blood test accurately detects lymphedema, study shows

Dec 18, 2012

Scientists at the Stanford University School of Medicine have identified a set of proteins circulating in blood whose levels accurately flag the presence of lymphedema. The findings, to be reported Dec. 18 in PLoS ONE, spur optimism that this common but relatively neglected condition, which affects an estimated 10 million people in the United States, finally will be amenable to detection (and, eventually, treatment) with 21st-century techniques.

Lymphedema is an often-painful inflammatory condition resulting from the blockage of lymphatic vessels that ordinarily drain fluid from the tissues throughout the body. In the developed world, lymphedema most often arises as an unintended consequence of radiation therapy for cancer. For example, about one in four breast-cancer survivors eventually develops lymphedema, said Stanley Rockson, MD, professor of cardiovascular medicine and the study's senior author. Numerous other factors, including parasitic infections endemic in some developing countries, can cause it as well, he said. 

The blunting of normal immune-cell flow due to lymphatic-vessel blockage helps to trigger the buildup of fluid within the affected area of the body, along with thickening of the skin, profound inflammation, accumulation of fibrous tissue, excessive blood-vessel formation and a marked expansion of the fatty layer beneath the skin. 

By the time the main symptom—swelling of one or more limbs—is detectable, the condition may have gotten such a foothold that it becomes difficult or impossible to reverse, at least given current treatment options, Rockson said. 

The only known way to diagnose lymphedema now is via physical inspection, and all too often it is misdiagnosed or overlooked altogether. But the biological events underpinning this condition may be present five years or more before symptoms become evident, said Rockson. Moreover, there are no effective drugs for combating lymphedema, just costly, time-consuming and annoying physical therapy, which virtually never completely eliminates the symptoms. While physical therapy can arrest progression and reduce swelling by as much as half, the condition typically remains a long-term problem. "Lymphedema virtually never just goes away on its own," said Rockson. Indeed, it tends to progress in severity over time, whether it is treated or not.

The irreversible skin thickening, joint immobility, scarring, increased susceptibility to infection and other consequences of chronic lymphedema can leave patients discomfited for life, all too often resulting in social withdrawal, body-image deterioration and other quality-of-life issues. "This is especially ironic in the case of cancer survivors who have endured difficult life-saving interventions, only to find that now cured, they're unable to enjoy their lives," said Rockson, who is the Allan and Tina Neill Professor of Lymphatic Research and Medicine and chief of consultative cardiology at the medical school. 

For this study, Rockson and his associates obtained skin-biopsy samples from both lymphedematous and normal tissue of 27 patients. Using advanced molecular methods, they compared each patient's diseased tissue with that same patient's healthy tissue to see which genes—the recipes for the myriad proteins produced in our bodies—were more actively engaged in the generation of their respective protein products in diseased versus healthy tissue. Thousands of genes fit the bill. Then the investigators narrowed their search to the overproduced proteins themselves, in particular ones that were already known to circulate throughout the bloodstream of all people, including healthy ones, and for which fast, commercial blood tests already exist. 

Statistical modeling indicated a panel of tests that measured six separate proteins' levels in study subjects' blood was able to distinguish the lymphedematous patients from control subjects who did not have lymphedema. None of these six proteins was predictive by itself. But in aggregate, their presence at certain levels and ratios appeared to serve as a biological fingerprint, or biomarker, for lymphedema. 

Interestingly, all six proteins are well-known, and each is associated with one or another of chronic lymphedema's hallmark biological features: accumulation of fibrous deposits, stimulation of fat-cell activity, inflammation and lymphatic-vessel formation and repair. "These biomarkers may themselves lead us to valuable pharmaceutical targets," said Rockson. 

To determine their six-protein biomarker-panel's validity, Rockson's group collected blood from a new cohort of 36 lymphedematous and 15 healthy adults, extracted blood samples and tested them with the panel. The test distinguished those with lymphedema from healthy subjects with an accuracy approaching 90 percent—good enough for use as a clinical 

diagnostic tool and a vast improvement over current detection methods, said Rockson. 

"This is a significant development," he said. 

Because levels of the six proteins begin to climb early in the course of the disease, such a test should be valuable in determining risk for, or the onset of, lymphedema long before symptoms occur—which in turn would mean earlier, appropriate therapeutic intervention, perhaps in time to spare patients from the condition's most-damaging effects or even reverse its course. "In addition," Rockson said, "a standardized, accurate bioassay for lymphedema could help to pave the road for future human clinical trials of drugs to treat it." Monitoring trial subjects at the molecular level with a lymphedema-detecting blood test could provide early evidence regarding whether an experimental treatment is working. Rockson is involved in conducting clinical trials of pharmaceutical agents for lymphedema, and hopes to use the new test in those trials. Journal reference: PLoS ONE Provided by Stanford University Medical Center 

Medical express

The published study itself:

PLoS One

Massive localized lymphedema of the male external genitalia: a clinicopathologic study of 6 cases.

Massive localized lymphedema of the male external genitalia: a clinicopathologic study of 6 cases.

Sept 2012

Lee S, Han JS, Ross HM, Epstein JI.

Source

Department of Pathology, The Johns Hopkins Hospital, Baltimore, MD 21287, USA.

Abstract

Massive localized lymphedema is a reactive pseudotumor strongly associated with obesity. The tumor most commonly presents as pendulous masses in the lower limbs with only 3 reported cases involving external male genitalia. In this study, we report an additional 6 cases localized to the external male genitalia. The cases were retrospectively identified from the surgical pathology database of the Johns Hopkins Hospital. All 6 patients were obese (5 presented with diffuse scrotal edema and 1 with a penile mass). 

In all cases, the clinical impression was of a benign chronic process developing over 3 months to 1 year. All 3 cases from outside institutions were referred with benign pathologic diagnoses. The lesions ranged in size from 4 to 55 cm. Microscopically, all cases exhibited stromal fibrosis and edema, multinucleated stromal cells, perivascular chronic inflammation, and lymphangiectasia. Entrapped fat was a minor feature and seen in only 3 cases. Variable hyperplasia and hypertrophy of dartos muscle were noted in 6 lesions. Three cases showed prominent microvascular proliferation around the edge of individual dartos muscle bundles. 

In summary, diagnosis of massive localized lymphedema requires appropriate correlation between clinical and microscopic findings. Lesions in the male external genitalia share many microscopic findings with massive localized lymphedema at other sites, although entrapped adipose tissue is not prominent. Additional, although not specific, findings include variably hyperplastic and hypertrophic dartos muscle and capillary neoangiogenesis at the interface between smooth muscle bundles and stroma.

PubMed

see also: Male Genital Lymphedema

The Lymphatics and Inflammation

The primary valves in the initial lymphatics during inflammation.

"More substantiation on the involvment of lymphedema and the body's inflammatory response system."

Lymphat Res Biol. 2007

Lynch PM, Delano FA, Schmid-Schönbein GW. Department of Bioengineering, University of California San Diego, La Jolla, California 92093-0412, USA. g...@bioeng.ucsd.edu

BACKGROUND: The primary valve system in the initial lymphatics prevents fluid transport from the initial lymphatics back into the interstitium. The authors hypothesize that since the primary valves are made up of an extraordinarily thin endothelium, they are readily compromised by mechanical or biochemical inflammatory stimuli. Thus, the opening dimension of the primary valves and their ability to prevent reflux into the interstitium during inflammation were investigated.

METHODS AND RESULTS: Acute inflammation was generated in the intact rat spinotrapezius muscle by suffusion of f-Met-Leu-Phe and platelet-activating factor. Once inflamed, the effective opening dimensions of the primary valves and the transport back out of the initial lymphatics were determined by examining the transport of fluorescent tracers from the interstitium to the lymphatics. Quantum dots and fluorescently labeled albumin readily enter initial lymphatics from the interstitium. The maximum diameter of microspheres that enter the initial lymphatics is between 0.5 microm and 0.8 microm in both control and inflamed tissue. While under control conditions no quantum dots escaped from initial lymphatics back into the interstitium, during inflammation there was extensive escape of quantum dots.

CONCLUSIONS: These results suggest that, in acute inflammation, the function of the endothelial barriers in the initial lymphatics may be compromised. A failure of the primary lymphatic valves has two consequences. First, fluid clearance from the tissue is less efficient, which causes the level of edema to increase. Second, the leaking initial lymphatics allow inflammatory mediators to accumulate in the tissue, therefore enhancing interstitial and lymphatic inflammatory reactions.

Article

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Inflammation, lymphatic function, and dendritic cell migration.

Lymphat Res Biol. 2006

Angeli V, Randolph GJ. Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York, USA. micva@nus.edu.sg

The lymphatic system is not only essential for maintenance of normal fluid balance, but also for proper immunologic function by providing an extensive network of vessels, important for cell trafficking and antigen delivery, as well as an exclusive environment, the lymph node (LN), where antigen-presenting cells (APCs) and lymphocytes can encounter and interact. Among APCs, dendritic cells (DCs) have a remarkable capacity to traffic from peripheral tissues to the draining LN, which is critical for execution of their functions.

To reach the LN, DCs must migrate towards and enter lymphatic vessels. Here, the authors review what is known about the factors that drive this process. They touch particularly on the topic of how DC migration is affected by inflammation and discuss this in the context of lymphatic function.

Traditionally, inflammatory mediators are regarded to support DC migration to LNs because they induce molecules on DCs known to guide them to lymphatics. The authors recently showed that inflammatory signals present in a strong vaccine adjuvant induce swelling in LNs accompanied by lymphangiogenesis in the draining LN and radius of peripheral tissue. These increased lymphatics, at least for several days, lead to a more robust migration of DCs.

However, the density of lymphatic vessels can become overly extended and/or their function impaired as observed during lymphedema and various chronic inflammatory reactions. Diseases characterized by chronic inflammation often present with impaired DC migration and adaptive immunity. Gaining a better understanding of how lymphatic vessel function may impact adaptive immunity by, for example, altering DC migration will benefit clinical research aiming to manipulate immune responses and manage chronic inflammatory diseases.

Article

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Inflammatory manifestations of experimental lymphatic insufficiency.

PLoS Med. 2006 Jul

Tabibiazar R, Cheung L, Han J, Swanson J, Beilhack A, An A, Dadras SS, Rockson N, Joshi S, Wagner R, Rockson SG. Stanford Center for Lymphatic and Venous Disorders, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California, United States of America.

BACKGROUND: Sustained lymph stagnation engenders a pathological response that is complex and not well characterized. Tissue inflammation in lymphedema may reflect either an active or passive consequence of impaired immune traffic.

METHODS AND FINDINGS: We studied an experimental model of acute post-surgical lymphedema in the tails of female hairless, immunocompetent SKH-1 mice. We performed in vivo imaging of impaired immune traffic in experimental, murine acquired lymphatic insufficiency. We demonstrated impaired mobilization of immunocompetent cells from the lymphedematous region. These findings correlated with histopathological alterations and large-scale transcriptional profiling results. We found intense inflammatory changes in the dermis and the subdermis. The molecular pattern in the RNA extracted from the whole tissue was dominated by the upregulation of genes related to acute inflammation, immune response, complement activation, wound healing, fibrosis, and oxidative stress response.

CONCLUSIONS: We have characterized a mouse model of acute, acquired lymphedema using in vivo functional imaging and histopathological correlation. The model closely simulates the volume response, histopathology, and lymphoscintigraphic characteristics of human acquired lymphedema, and the response is accompanied by an increase in the number and size of microlymphatic structures in the lymphedematous cutaneous tissues. Molecular characterization through clustering of genes with known functions provides insights into processes and signaling pathways that compose the acute tissue response to lymph stagnation. Further study of genes identified through this effort will continue to elucidate the molecular mechanisms and lead to potential therapeutic strategies for lymphatic vascular insufficiency.

Article