Current views on the function of the lymphatic vasculature in health and disease - 나중에 다시 정리

[문형철]

항암치료, 장기간 inactive 환자, 장기 immobilization환자의 순환장애를 효과적으로 개선하기 위한 핵심이 있다. 

동맥혈순환

정맥혈순환

림프순환

swelling과 edema 등의 구분을 위해서

림프 순환에 대한 이해를 위하여

격렬한 움직임을 통해 근육의 혈액공급량은 800배까지 증가한다. 

panic bird...

lymphatic vascular system의 세가지 필수 기능

1. lipid absorption

2. fluid homeostasis

3. immune surveillance

질병

lymphedema(림프부종)

Current views on the function of the lymphatic vasculature in health and disease.pdf

The lymphatic vascular system is essential for lipid absorption, fluid homeostasis, and immune surveillance. Until recently, lymphatic vessel dysfunction had been associated with symptomatic pathologic conditions such as lymphedema. Work in the last few years had led to a better understanding of the functional roles of this vascular

system in health and disease. 

임파혈관계는 지질흡수, 체액항상성, 면역감시기능에 필수적 역할을 수행함.

최근까지 임파혈관계 기능부전은 림프부종과 같은 질병과 연관되어 있었음.

지난 몇년간 임파혈관계의 기능적 역할에 대한 이해가 축적되어 옴. 

Furthermore, recent work has also unraveled additional functional roles of the lymphatic vasculature in fat metabolism, obesity, inflammation, and the regulation of salt storage in hypertension. In this review, we summarize the functional roles of the lymphatic vasculature in health and disease.

최신의 연구에 의하면 지질대사, 비만, 염증, 고혈압에서 소금저장의 조절 기능에 대한 탐구가 이어짐. 

이 논문에서 우리는 임파혈관계의 기능적 역할에 대해서 정리할 것임. 

In mammals, two specialized vascular systems are responsible for effective circulation: the blood vasculature, which delivers oxygen and nutrients, and the lymphatic vasculature, which transports fluid and macromolecules from tissues back to the blood circulation. These two highly branched endothelial tubular networks are closely related. In mammals, lymphatic vessels originate from embryonic veins and forman independent, unidirectional vascular system that interconnects with blood vessels at a few specific sites (Karpanen and Alitalo 2008). 

동물은 두가지 특별한 혈관계가 순환을 담당함

혈관구조는 산소와 영양분을 운반하고, 임파맥관구조는 혈장과 거대분자를 조직으로부터 혈액순환으로 이동하는 역할을 수행함. 

이 두가지 내피관모양의 네트워크 가지는 서로 밀접하게 연관됨. 

동물에서 임파관(lymphatic vessel)은 배아정맥으로부터 유래하고, 구멍 독립적이고 단일방향성의 혈관시스템은 몇군데 특별한 부위에서 혈관과 서로 연결됨. 

The appropriate function of blood vessels is crucial for the delivery of oxygen and nutrients and carries away waste products for detoxification and replenishment. The lymphatic vasculature, however, plays important roles in

immune surveillance, lipid absorption, and maintenance of tissue fluid balance (Harvey and Oliver 2004; Oliver and Alitalo 2005; Tammela and Alitalo 2010).

혈관의 중요한 기능은 산소와 영양분을 운반하고 해독과 보충을 위해 찌꺼기 부산물을 운반함. 

임파맥관구조는 면역기능, 지질흡수, 조직혈장균형 유지에 중요한 역할을 수행함. 

The lymphatic system is also crucial during the immune response to infectious agents because afferent lymphatic vessels are the route through which dendritic cells migrate to the lymph nodes and lymphoid organs after antigen uptake. Dendritic cells, macrophages, and memory T lymphocytes use the lymphatic network to move from peripheral tissues to regional lymph nodes. 

임파시스템은 또한 감염물질에 면역반을을 하는동안 중요한 역할을 함. 항원이 들어온 후 임파절과 임파기관으로 dendritic 세포가 이동하는 통로가 되는 구심성 임파혈관이기 때문에..

수지상세포, 대식세포, 기억 t 임파세포는 임파네트워크를 이용하여 말초조직으로부터 각지의 임파절로 이동함. 

참고) dendritic cell

Dendritic cells (DCs) are antigen-presenting cells, (also known as accessory cells) of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.

Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymph nodes where they interact withT cells and B cells to initiate and shape the adaptive immune response. 

In most forms of cancer, metastastatic spreading of tumor cells occurs through the lymphatic vascular network. Although few life-threatening diseases result from malfunction of the lymphatic vasculature, failure of lymph transport promotes lymphedema, a disfiguring, disabling, and occasionally lifethreatening disorder.

대부분 암세포는 임파맥관 네트워크를 통하여 전이함. 

비록 생명을 위협하는 질환 몇가지가 임파맥관의 비정상으로 야기되지만, 림프이동의 실패는 림프부종을 촉진하여 흉칙한 모양을 만들고, 기능부전이 일어나고 가끔 생명을 위협하기도 함. 

In the last few years, a number of genes regulating different aspects of developmental and postnatal lymphangiogenesis

have been identified (Oliver and Srinivasan 2008) and valuable animal models have become available. These tools have not only helped us to improve our understanding of different lymphatic-related pathologic conditions and their relationship with inflammation, autoimmunity, and cancer, but also to re-eval‎uate the functional roles of the lymphatic vascular network. 

최근 몇년간의 연구는 임파혈관재생에 관하여 유전자 조절의 측면에서 진행됨. 이러한 연구는 염증, 자가면역, 암과 연관된 임파관련 병리에 대한 이해를 증진시켰을 뿐 아니라 lymphatic vascular network의 기능적 역할에 대해서 재조명함. 

For example, lymphatic vascular defects were identified as leading causes of late-onset obesity in a mouse model (Harvey

et al. 2005), and subcutaneous lymphatic vessels participate in regulating blood pressure after excessive salt intake (Machnik et al. 2009). 

늦은 발병 비만의 주요원인은 임파혈관구조 defect로 정의됨. 

피부 임파혈관은 과도한 소금섭취후에 혈압조절에 관여함. 

In this review, we focus on some of the recent progress in the field of lymphatic vasculature malfunction and diseases.

Lymphatic vasculature

During embryonic development, the blood vasculature is generated through two distinct processes known as  vasculogenesis and angiogenesis (Risau and Flamme 1995; Flamme et al. 1997). Vasculogenesis is the de novo assembly

of vessels by endothelial precursor cells, so-called angioblasts, and angiogenesis involves the growth and remodeling of pre-existing blood vessels into a more complex and elaborated vascular network (Adams and Alitalo 2007). 

배아형성기동안 혈관구조는 두가지 분명한 구조를 생성함. vasculogenesis와 angiogenesis

The development of the lymphatic vasculature is secondary to that of blood vessels. Using a detailed lineage tracing approach, Srinivasan et al. (2007) have recently confirmed the proposal made by Sabin (1902) more than a century ago postulating that the lymphatic vasculature originates from the embryonic veins.

임파혈관구조는 혈관에 이차적으로 발달함. 

The lymphatic vascular network develops in a stepwise manner (Oliver and Detmar 2002;Wigle et al. 2002; Oliver

2004). In the mouse, this process starts at approximately embryonic day 9.75 (E9.75) in the anterior cardinal vein

(Srinivasan et al. 2007). Some of the key players controlling  the necessary steps in this process have been identified in

the last decade (Oliver 2004; Harvey and Oliver 2004; Oliver and Alitalo 2005; Adams and Alitalo 2007).

The lymphatic vascular network consists of smaller blind-ended capillaries and larger collecting lymphatic vessels (Fig. 1). The lymphatic capillaries are composed of a single layer of overlapping endothelial cells (ECs) and lack a continuous basement membrane and pericytes (i.e., the smooth muscle-like contractile cells that wrap around the outer surface of blood vessels) (Fig. 1). 

임파 혈관네트워크는 작은 끝이 막힌 관구조이고 큰 임파혈관으로 모이는 구조임. 

임파 capillaries는 중첩된 내피세포의 단층으로 구성되고 ...

Therefore, the lymphatic capillaries are highly permeable to interstitial fluid and macromolecules, such that, when the surrounding interstitial pressure changes, these lymphatics either expand and fill with lymph or contract and push lymph

(Leak and Burke 1966). These lymphatic capillaries first drain into precollecting lymphatic vessels, which will eventually merge into larger secondary collecting lymphatic vessels covered by smooth muscle cells that provide contractile activity to assist lymph flow and possess a continuous basement membrane. Tissue fluid collected in the larger collecting lymphatics drains into the thoracic duct and is then returned to the blood circulation through lymphatic–vasculature connections at the junction of the jugular and subclavian veins.

주위 interstitial pressure가 변화할 때, 임파 capallaries는 interstitial 활액과 거대분자에 높은 투과성을 가지고 있음. 

이때 임파관에 림프액으로 가득차면서 확장되거나 림프액을 밀어올려 이동함. 

이러한 임파미세혈관은 임파관으로 림프액이 모이게함... 임파관을 둘러싸고 있는 smooth muscle cell의 수축에 의해서 림프액은 이동함. 임파액은 thoracic duct 에 모이고, 혈관으로 이어지는데 jugular and subclavian vein으로 연결됨..

참고)  thoracic duct and subclavian vein

In human anatomy, the thoracic duct is the largest lymphatic vessel of the lymphatic system. It is also known as the left lymphatic duct, alimentary duct, chyliferous duct, and Van Hoorne's canal.

In adults, the thoracic duct is typically 38-45cm in length and an average diameter of about 5mm. The vessel usually starts from the level of the second lumbar vertebra and extends to the root of the neck. It drains into the systemic (blood) circulation at the left subclavian vein. It also collects most of the lymph in the body other than from the right side which is drained by the right lymphatic duct.

The lymph transport, in the thoracic duct, is mainly caused by the action of breathing, aided by the duct's smooth muscle and by internal valves which prevent the lymph from flowing back down again. There are also two valves at the junction of the duct with the left subclavian vein, to prevent the flow of venous blood into the duct. In adults, the thoracic duct transports up to 4 L of lymph per day.

Lymphatic vasculature malfunction and disease Lymphedema

Malfunction of the lymphatic vasculature can result in congenital or acquired disorders such as lymphedema (i.e., imbalance in lymph absorption), which is a disfiguring and disabling disorder often characterized by swelling of the extremities, tissue fibrosis, accumulation of subcutaneous fat, and susceptibility to infections (Fig. 2; Rockson 2001; Witte et al. 2001; Johnson and Oliver 2009). 

Primary lymphedema has a genetic origin and secondary lymphedema arises as the result of surgery, infection (e.g., filiariasis), or radiation therapy (Rockson 2001; Witte et al. 2001). Primary lymphedema can be present at birth in the form of Milroy disease, an inherited form of primary lymphedema characterized by the absence or reduction in the number of lymphatic vessels (Milroy 1892), or appear after birth, mainly around puberty (Meige disease) (Meige 1898). In general, both of these diseases are characterized by dilated lymphatic vessels and accumulation of lymph fluid.

The typical accumulation of interstitial fluid observed in lymphedema results from insufficient lymph transport or occlusion of lymphatic drainage caused by hypoplasia or damage of the lymphatic vessels, impaired lymphatic function, or obstruction of lymph flow (Fig. 2; Witte et al. 2001). 

Although lymphedema is rarely a life-threatening condition, it severely affects the quality of life of affected individuals, and effective treatment is unavailable. Primary lymphedema Specific genetic defects have been identified recently in hereditary diseases associated with primary lymphedema. For example, heterozygous missense mutations in vascular endothelial growth factor receptor 3 (VEGFR3) have been identified in several cases of Milroy disease (Ferrell et al. 1998; Irrthum et al. 2000; Karkkainen et al. 2000). Mutations in this gene are also responsible for the mutant mouse strain Chy, which has defective lymphatic vessels and develops chylous ascites and lymphedematous limb swelling after birth (Karkkainen et al. 2001). This animal model has been used to test targeted gene therapy and adenoviral expression‎ of VEGF-C (one of the ligands of VEGFR3) (Joukov et al. 1996, 1997; Achen et al. 1998), which promotes the formation of functional lymphatic vessels in these mice (Karkkainen et al. 2001). 

Functional inactivation of Vegfc in mice revealed that this gene’s activity is essential for the budding and proliferation of lymphatic ECs (LECs) that are located in the embryonic veins and express Prox1, a transcription factor essential for specifying the LEC phenotype and forming the entire lymphatic vasculature network (Karkkainen et al. 2004;Wigle et al. 2002). However, Vegfc heterozygous mice survive to adulthood, but exhibit cutaneous lymphatic hypoplasia and lymphedema (Karkkainen et al. 2004).

Similarly, mutations in the forkhead transcription factor FOXC2 have been identified in patients with lymphedema-ditichiasis (LD) syndrome (Fang et al. 2000; Finegold et al. 2001). This autosomal dominant disorder is characterized by distichiasis (i.e., a double row of eyelashes) at birth and bilateral lower limb lymphedema at puberty (Neel and Schull 1954; Falls and Kertesz 1964). Unlike congenital lymphedema, the number of lymphatic vessels appears to be normal in individuals with LD; however, these patients have impaired lymphatic drainage (Brice et al. 2002). This alteration could be due to the abnormal mural cell coating of the lymphatic capillaries and the lack of luminal valves in the collecting lymphatics that has been identified in patients with LD and in Foxc2 mutant mice (Fig. 2; Petrova et al. 2004).

Mutations in the transcription factor SOX18 were identified in recessive and dominant forms of hypotrichosis-

lymphedema-telangiectasia (HLTS), a rare disease characterized by the absence of eyebrows and eyelashes, edema of the inferior members or eyelids, and peripheral vein anomalies (Irrthum et al. 2003). In mice, depending on the genetic background, the functional inactivation of Sox18 results in embryonic lethality, and the mutant embryos exhibit edema and lack a lymphatic vasculature (Francois et al. 2008). Sox18 was reported to act as an upstream regulator of Prox1 expression‎ in the embryonic anterior cardinal vein (Francois et al. 2008). The Ragged mouse, which has a spontaneous pointmutation in Sox18, is considered to be a likely model for HLTS. These mice exhibit defective vasculogenesis and folliculogenesis as well as malformation of lymphatic vessels, which are similar to those of humans with HLTS (Pennisi et al. 2000; Irrthum et al. 2003; Francois et al. 2008).

Another gene affected in primary lymphedema is integrin-a9 (ITGA9): Mutations in this gene have been reported recently in fetuses with congenital chylothorax (Ma et al. 2008). Similar to humans with this condition, Itga9-null mice exhibit chylothorax and die a few days after birth (Huang et al. 2000), and the characterization of recently generated Itga9-conditional mutant embryos has revealed that ITGA9 is required for proper lymphatic valve morphogenesis (Bazigou et al. 2009).

Hennekam syndrome is a rare, heritable disease characterized by lymphedema, lymphangiectasia, and developmental

delay; the lymphedema usually becomes apparent in the face and limbs at birth or in early infancy (Van Balkom et al. 2002). Mutations in collagen and calciumbinding EGF domain-1 (CCBE1) are responsible for this disease (Alders et al. 2009), and ccbe1 is required for embryonic lymphangiogenesis and lymphatic sprouting from the venous endothelium in zebrafish (Hogan et al. 2009).

Unexpectedly, mutations in RELN, a gene thought to function only in the brain, have also lead to congenital lymphedema

in at least three patients with persistent neonatal lymphedema, and in one with accumulation of chyle (Hong et al. 2000).

Finally, although the chromosomal locations for Turner syndrome (whose patients exhibit lymphedema) and cholestasis-lymphedema (i.e., Aagenaes syndrome) have been mapped to Xp11.2–p22.1 and 15q, respectively, the identity of the genes involved in these disorders remains unknown (Zinn et al. 1998; Bull et al. 2000). Secondary lymphedema Worldwide, most cases of lymphedema are secondary and due to some type of damage to the lymphatic vasculature. Among the causes of this damage, filariasis (i.e., elephantiasis) is the most common one, affecting >100 million people, mostly in tropical areas (Wynd et al. 2007; Pfarr et al. 2009). Filariasis itself is the result of parasitic infection by mosquito-borne worm parasites that localize to the lymphatic system, where an inflammatory reaction triggers the production of VEGF, VEGF-C, and VEGF-D. Eventually, hyperplasia, obstruction, and extensive damage of the lymphatic vasculature occur (Fig. 2), resulting in chronic lymphedema of the lower limbs or genitalia and permanent disability (Rockson 2001; Pfarr et al. 2009). 

Treatment with drugs targeting the microfilariae (larval offspring) has been the main approach to treat this disease (Hoerauf et al. 2003). Recently, a novel more efficient approach aimed to directly target the intracellular bacterial symbiont of filarial parasites (Wolbachia) was shown to kill most adult worms without causing severe side effects (Stolk et al. 2005). In contrast to the abundance of filariasis-promoted lymphedema in tropical areas, the leading cause of secondary lymphedema in the industrialized world is lymph node dissection or radiation therapy that damages the lymphatic vasculature (Fig. 2) after breast cancer surgery (Rockson 2001) and affects ;15%–20% of women undergoing breast cancer treatment (Vignes et al. 2007).

Unfortunately, treatment for lymphedema is still based mainly on conservative therapies such asmanual drainage, massage, compression garments, liposuction, and dietary

modification (i.e., limiting the consumption of long-chain

fatty acids) (Rockson 2001; Brorson 2003). Previous work

has shown that VEGF-C can induce capillary lymphatic

vessel growth in animal models (Karkkainen et al. 2001;

Szuba et al. 2002; Yoon et al. 2003; Saaristo et al. 2004), and a recent study in mice demonstrated that treatment with

adenovirus-delivered VEGF-C/D promotes the regeneration

of functional collecting lymphatic vessels after the

excision of axillary lymph nodes and the associated

collecting lymphatic vessels (Tammela et al. 2007). Therefore,

it appears that VEGF-C/D growth factor therapy combined

with lymph node transplantation could become

a feasible clinical approach to restore the entire lymphatic

network in damaged tissues, particularly in cases

of injury or side effects following surgery (Tammela et al.

2007). 

Lymphangioleiomyomatosis (LAM)

LAM is a rare, cystic lung disease that is progressive and

often lethal (Kumasaka et al. 2004, 2005; Juvet et al. 2006,

2007; Seyama et al. 2006). LAM is characterized by the

proliferation of abnormal smooth muscle-like cells (i.e.,

LAMcells) that formLAMlesions in the lungs, axial lymph

nodes, and other organs. This disease is often associated

with renal angiomyolipomas (AMLs) (Kumasaka et al.

2004, 2005; Juvet et al. 2006, 2007; Seyama et al. 2006)

and frequently causes chylothorax by obstructing pulmonary

lymphatic vessels (Kumasaka et al. 2004; Juvet et al.

2007). LAM primarily affects females of childbearing age

and results from mutations in one of the tuberous sclerosis

complex (TSC) genes, usually TSC2 (Kumasaka et al. 2005;

Karpanen and Alitalo 2008). Interestingly, it has been

proposed that LECs play an essential role in LAM lesion

dissemination, and that LAM-associated lymphangiogenesis plays a role in LAM progression (Kumasaka et al. 2004, 2005). Recent results have revealed that VEGF-C and VEGF-D induce the proliferation of LAM-derived cells (LDC) via an autocrine cross-talk with LECs (Issaka et al.2009).

Lymphangiectasia

Lymphangiectasia (i.e., dilation of the lymphatic vessels) is most often seen in the lung, intestine, and thoracic cavity (Faul et al. 2000). Congenital pulmonary lymphangiectasia is a rare disorder of newborns and is often fatal. Affected individuals exhibit cyanosis and labored breathing, and often have chylothorax or chylous effluence in the thoracic cavity (Bellini et al. 2006). Intestinal lymphangiectasia is characterized by highly dilated lymphatic capillaries in the intestinal villi. Because the normal lymphatic response to changes in interstitial pressure is hindered by this hyperdilation, absorption by the intestine is compromised.

Moreover, the levels of FOXC2 and SOX18 transcription are significantly lower in these patients (Hokari et al. 2008; Johnson and Oliver 2009).

Lymphatic vasculature and inflammation

Inflammation is the normal biological response of vascular

tissues to harmful stimuli such as injury, infection, or

tumors. Although it is well known that the blood vasculature

is an important regulator of the inflammatory process,

the role of the lymphatic network in this process is

becoming better understood, as several lines of new evidence

revealed that inflammation triggers lymphangiogenic

signals (Cursiefen et al. 2004; Kunstfeld et al. 2004;

Baluk et al. 2005, 2009; Maruyama et al. 2005; Kerjaschki

et al. 2006; Flister et al. 2010). Additionally, blood and

lymphatic vessels undergo extensive remodeling during

chronic inflammation (e.g., asthma, pulmonary disease,

rheumatoid arthritis, and inflammatory bowel disease).

During inflammation, different chemokine receptors

regulate the recruitment of leukocytes and the inflammatory

response. Among those, the chemokine receptor

D6 acts as a decoy for inflammatory CC chemokines, and

mice deficient for this receptor exhibited increased inflammation

(Jamieson et al. 2005; Martinez de la Torre

et al. 2005). Interestingly, this receptor is expressed on the

lymphatic endothelium, where it might influence the

inflammatory response (Nibbs et al. 2001).

The lymphatic vasculature regulates inflammatory responses

by transporting leukocytes and antigen-presenting

cells fromthe site of inflammation to the lymph nodes and

other secondary lymphoid organs. After inflammatory reactions,

activated dendritic, T, and B cells capture antigens

and migrate through afferent lymphatic vessels into the

lymph nodes. At the same time, macrophages and mast

cells are activated, and residential and recruitedmonocytederived

macrophages are crucial for evoking and resolving

the acute inflammatory reaction. Dendritic cell mobilization

is stimulated by VEGF produced by B cells in inflamed

lymph nodes (Angeli et al. 2006), and inflamed lymphatic

endothelium can suppress dendritic cellmaturation through

aMac-1/ICAM-1-dependentmechanism (Podgrabinska et al.

2009).

Lymphangiogenesis occurs at sites of tissue inflammation, and macrophages express VEGF-C and VEGF-D, which contribute to lymphatic vessel formation during

inflammation (Cursiefen et al. 2004;Maruyama et al. 2005;

Kerjaschki et al. 2006). For example, lymphangiogenesis is

associated with inflammatory angiogenesis in the rabbit

cornea, and the angiogenic and lymphangiogenic responses

are blocked by depleting macrophages, a result suggesting

that inflammatory cells mediate lymphatic vessel formation

(Cursiefen et al. 2004). In fact, CD11b+ macrophages

are involved in inflammation-induced lymphangiogenic

growth in the cornea (Maruyama et al. 2005). Furthermore,

during inflammatory conditions such as penetrating keratoplasty

in the mouse cornea, CD11b+ macrophages can

transdifferentiate into LECs and contribute to lymphangiogenesis

in the corneal stroma (Maruyama et al. 2005).

However, no evidence is yet available supporting a role for

macrophages during developmental lymphangiogenesis

or normal noninflammatory conditions (Srinivasan et al.

2007; Bertozzi et al. 2010).

Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. Extensive lymphangiogenesis promoted by VEGFC- and VEGF-D-producing inflammatory cells was observed in a mouse model of chronic airway inflammation induced by Mycoplasma pulmonis infection (Baluk et al.

2005). In this model, inhibiting VEGFR3-mediated signaling

prevented the growth of lymphatic vessels, leading to bronchial lymphedema and airflow obstruction (Baluk

et al. 2005). Therefore, it is argued that defective lymphangogenesis

during airway inflammation may lead to bronchial

lymphedema and exaggerated airflow stagnation

(Baluk et al. 2005). In the same animal model, airway

inflammation leads to increased tumor necrosis factor a

(TNF-a) expression‎, and inhibiting TNF-a signaling results

in significantly reduced lymphangiogenesis (Baluk

et al. 2009). Therefore, TNF-a might require inflammatory

mediators from recruited leukocytes to fulfill its role

in inflammation-mediated lymphangiogenesis (Baluk

et al. 2009).

Lymphangiogenesis was also reported in cases of chronic

inflammation such as psoriasis and rheumatoid arthritis

(Kunstfeld et al. 2004; Kajiya and Detmar 2006; Tammela

and Alitalo 2010), and in cases of kidney transplant rejection

in which the newly formed lymphatic vessels produce

the chemokine CCL21, which attracts CCR7-positive

dendritic cells that might promote the inflammatory reaction

(Kerjaschki et al. 2004). Consistent with the antiinflammatory

role of transforming growth factor b (TGF-b),

recent results revealed that TGF-b signaling acts as a

lymphangiogenesis inhibitor in inflammatory settings

(Clavin et al. 2008; Oka et al. 2008).

At the molecular level, the main mediators of the

inflammatory response are transcription factors of the

nuclear factor-kB (NF-kB) family. NF-kB is constitutively

active in the lymphatic vasculature (Saban et al. 2004), and

in vitro analysis suggests that induction of the NF-kB pathway

by inflammatory stimuli activates the transcription

factor Prox1, which acts with NF-kB to activate VEGFR3

(Flister et al. 2010). Prox1 (a key regulator in the pathway

leading to LEC specification) and VEGF-C (the VEGFR3

ligand) are both required to form the lymphatic network

(Wigle and Oliver 1999;Wigle et al. 2002; Karkkainen et al.

2004). In an inflammatory setting, the elevated VEGFR3

expression‎ promoted by the induction of NF-kB signaling

increases the sensitivity of the pre-existing lymphatic

endothelium to VEGF-C and VEGF-D, leading to enhanced

lymphangiogenesis (Flister et al. 2010). Furthermore, in

cultured T cells, interferon g, a major proinflammatory

effector, is repressed by Prox1 in cooperation with peroxisome

proliferator-activated receptor g (Wang et al. 2008).

Together, these findings revealed a series of novel mechanisms

underlying inflammatory lymphangiogenesis that

should be relevant in therapeutic approaches aimed at

eliminating the rejection reaction after organ transplantation

and treating inflammatory airway diseases.

Lymphatic vasculature and tumor metastasis

In most human cancers, the lymphatic vasculature serves

as the primary route for the metastatic spread of tumor

cells to regional lymph nodes (Van den Eynden et al. 2007;

Bolenz et al. 2009; Gao et al. 2009; Kodama et al. 2010).

Additionally, metastatic node status represents a major criterion for staging, treatment, and prognosis for many types of solid tumors. However, despite the importance of tumor-associated lymphatic vessels for cancer progression, not much information is yet available about the cellular and molecular mechanisms involved in lymphatic metastasis.

Tumor cells access the lymphatic vasculature by invading

the pre-existing lymphatic vessels in the tumor

margin or promoting lymphangiogenesis (Achen et al.

2005; Tobler and Detmar 2006). In some types of cancer,

malignant tumor cells may have an active role in inducing

peritumoral and intratumoral lymphangiogenesis

(He et al. 2005; Hoshida et al. 2006).

Several studies have shown that VEGF-C/D expression‎

facilitates tumor-associated lymphangiogenesis and promotes

lymph node metastasis (Karpanen et al. 2001;

Mandriota et al. 2001; Skobe et al. 2001; Stacker et al.

2001; Arigami et al. 2009; Lahat et al. 2009; Sugiura et al.

2009). Blocking VEGF-D or VEGF-C to VEGFR3 signaling

inhibits both tumor-associated lymphangiogenesis and

lymph node metastasis (Fig. 3; Karpanen et al. 2001;

Mandriota et al. 2001; Stacker et al. 2001; He et al. 2005;

Lin et al. 2005; Hoshida et al. 2006; Roberts et al. 2006). In

line with these results, expressing siRNA targeted against

VEGF-C in mouse breast cancer cells inhibits lymph node

metastasis (Chen et al. 2005; Shibata et al. 2008). In

addition, blocking the binding of VEGF-C to its coreceptor,

neuropilin-2 (Nrp2), reduces VEGF-C-induced LEC migration

aswell as tumor-associated lymphangiogenesis (Caunt

et al. 2008).

Recent results revealed that lymphangiogenesis is not

only involved in lymph node metastasis, but also contributes

to distant metastasis. For example, in the case of

lung tumors, work by Das et al. (2010) has shown recently

that, in addition to its effects in the primary tumor site

and the lymph nodes, VEGF-C activity in the lung tissue

also impact the metastatic progression. These data indicate

that VEGF-C–VEGFR3 signaling may be a potential

target for the treatment of metastatic tumors. Several independent

studies have shown that lymph node lymphangiogenesis

may promote tumor metastasis (Dadras et al. 2005; Hirakawa et al. 2005, 2007, 2009; Harrell et al. 2007; Van den Eynden et al. 2007). Interestingly, it appears that tumors can induce lymph node lymphangiogenesis at a distance, as tumor-associated lymphangiogenesis in lymph nodes starts even prior to tumor dissemination (Fig. 3; Hirakawa et al. 2005, 2007; Harrell et al. 2007). However, a possible role of lymph node lymphangiogenesis in facilitating distant tumor metastasis still needs to be determined.

There is an increasing amount of evidence highlighting

the involvement of the CC chemokine receptor 7 (CCR7)

in tumor metastasis (Muller et al. 2001; Wiley et al. 2001;

Issa et al. 2009). It has been shown recently that tumor

cells can produce both VEGF-C and CCR7, which act

cooperatively to promote lymphatic invasion (Issa et al.

2009). In addition to inducing lymphangiogenesis, tumorsecreted

VEGF-C up-regulates the expression‎ of CCL21, the

ligand of CCR7, in LECs to attract CCR7-expressing tumor

cells to lymphatic vessels. VEGF-C can also promote proteolytic

activity and motility of tumor cells, and, therefore,

enhance tumor cell invasion (Issa et al. 2009). Moreover,

tumor-associated macrophages (TAMs) in peritumoral

stroma produce VEGF-C and VEGF-D, contributing to peritumoral lymphangiogenesis and tumor metastasis

(Schoppmann et al. 2002). In addition, Kubota et al. (2009)

have demonstrated that macrophage colony-stimulating

factor (M-CSF), a cytokine required for monocyte lineage

cell differentiation, promotes the formation of high-density

vessels in tumors (Kubota et al. 2009). Inhibiting M-CSF

specifically suppresses tumor-associated angiogenesis and

lymphangiogenesis, suggesting that M-CSF might be a target

for anti-cancer treatment (Kubota et al. 2009).

Although peritumoral lymphatic vessels are functional,

the presence and significance of lymphatic vessels within

tumors is still controversial. For example, it has been

argued that, because of high intratumoral pressure, intratumoral

lymphatics might not be functional or required for

lymphatic metastasis (Jain 1989; Padera et al. 2002; Wong

et al. 2005). However, others have found that intratumoral

lymphatic vessel density is significantly associated with

lymphatic invasion (Kyzas et al. 2005).

Lymphatic vasculature and Kaposi’s sarcoma (KS)

KS is the most common malignant tumor in AIDS

patients infected with human immunodeficiency virus

(HIV) (Hong et al. 2004; Wang et al. 2004). KS is a highly

vascularized tumor whose formation requires infection

with KS-associated herpesvirus (KSHV, also known as

human herpesvirus-8). In KS tumors, spindle cells (i.e.,

cells of endothelial origin) are themost common cells, and

they were shown to express the LEC markers podoplanin

and VEGFR3 (Jussila et al. 1998; Skobe et al. 1999; Carroll

et al. 2004). Infecting blood vascular ECs (BECs) with

KSHV is sufficient to reprogram them into LECs in vitro

(Carroll et al. 2004; Hong et al. 2004; Wang et al. 2004). In

addition, it was shown previously that Prox1 is sufficient

to reprogramcultured BECs into LECs by repressing ;40%

of BEC-specific genes and inducing the expression‎ of;20%

of LEC-specific genes (Hong et al. 2002; Petrova et al. 2002).

In line with those findings, BEC-to-LEC reprogramming

promoted by KSHV infection is associated with up-regulated

Prox1 expression‎ in infected cells, and inactivating

Prox1 blocks this reprogramming (Hong et al. 2004). These

results suggest that KSHV infection of BECs leads to their

reprogramming into LECs as they transform into spindle

cells (Carroll et al. 2004). Increased serum levels of the

lymphangiogenic factors VEGF-D and angiopoietin-2 have

been detected in patients with AIDS and KS (Wang et al.

2004). As an extension of this initial work, Dadras et al.

(2008) demonstrated recently that Prox1 expression‎ is

sufficient to induce more aggressive behavior of KS tumors

growing in syngenic mice; this observation was associated

with up-regulation in the expression‎ of genes involved in

proteolysis, cell adhesion, and migration.

Novel functions of the lymphatic vasculature

Fat metabolism

The lymphatic vasculature is essential for the adsorption

of lipids from the intestine. In the 17th century, Aselli

(1627) observed the mesenteric lymphatics (structures

that he named the venae alba et lacteae; i.e., white veins)

in dogs who had consumed a lipid-rich meal. The lipidrich

content of the mesenteric lymphatics (lacteals) is

drained via the cisterna chyli and thoracic duct back into

the bloodstream. The lipid-rich intestinal content is

packaged by the absorptive cells of the small intestine

(i.e., enterocytes) into water-soluble particles named

chylomicrons.

The fact that, for example, lymph nodes are normally

surrounded by adipose tissue and subcutaneous adipose

tissue lies in close proximity to the dermal lymphatic

vasculature suggested a relationship between lymphatic vessels and adipose metabolism. Additionally, ectopic adipose

tissue growth is observed in edematous regions of

patients suffering from chronic lymphedema (Brorson 2003),

and lymph or chyle (i.e., a mixture of lymph and chylomicrons

with a milky appearance) promotes the differentiation

of rabbit preadipocytes in vitro (Nougues et al. 1988).

Furthermore, a correlation between immune cells, lymph

nodes, and their surrounding adipose tissue has been documented

(Pond and Mattacks 1995, 2003; Pond 2003). In rats,

chronic inflammation of the peripheral lymph nodes increases

the number of adipocytes surrounding the nodes

(Mattacks et al. 2003). Additional work by Mattacks et al.

(2003) demonstrated that adipocytes within the lymph node

fat pads that are most closely apposed to the lymph node

respond to local immune challenge by increasing their rate

of lipolysis.

In the case of human pathologies, lipedema is a chronic

syndome that arises almost exclusively in post-pubertal

females and causes characteristic swelling of the bilateral

and symmetric lower extremities as a consequence of

excess subcutaneous fat deposition, although the feet

remain normal (Rudkin and Miller 1994; Bilancini et al.

1995; Fonder et al. 2007; Harvey 2008; Suga et al. 2009).

Defective lymphatic vasculature should not be ruled out

as a leading cause of this disease because functional alterations

in lymph flow (Bilancini et al. 1995) and microlymphatic

aneurysms (Amann-Vesti et al. 2001) have been

described in the skin of patients with lipedema.

Despite all of these well-established connections between

lymphatics and lipids, the role of the lymphatic

vasculature in adiposemetabolismhas only recently begun

to be recognized.

Increased deposition of subcutaneous fat in edematous

regions has been observed in Chy mice carrying a heterozygous

inactivating mutation in VEGFR3 (Karkkainen

et al. 2001). These mice exhibit lymphedema as a result of

hypoplastic cutaneous lymphatic vessels (Karkkainen

et al. 2001). This finding supports the hypothesis that

the lymph is adipogenic.

A correlation between late-onset obesity and the lymphatic

vasculature has been demonstrated in a mouse

model of defective lymphatic vasculature providing the

strongest direct evidence of a link between lymphatics

and fat metabolism. In this model, Harvey et al. (2005)

demonstrated that Prox1 haploinsufficiency promotes

lymphatic vascular defects that cause adult-onset obesity

(Fig. 2); therefore, Prox1 heterozygous mice serve as

the first in vivo model of lymphatic-mediated obesity

(Harvey et al. 2005; Harvey 2008). The leading cause of

the obese phenotype resulting from Prox1 haploinsufficiency

is abnormal lymph leakage due to a disruption in

lymphatic vascular integrity, particularly of the mesenteric

lymphatic vessels (Fig. 2; Harvey et al. 2005). Furthermore,

the lymph was capable of promoting adipogenic

differentiation of mouse 3T3-L1 preadipocytes, leading to

the proposal that disruption of lymphatic vascular integrity

promotes the ectopic growth of fat in lymphaticrich

regions because of increased lipid storage in adipocytes

and increased differentiation of preadipocytes to

mature adipocytes (Harvey et al. 2005).

In Prox1+/ mice, adipose tissue accumulation is associated

with an increased number of LYVE-1-positive macrophages

in the mesentery (Harvey et al. 2005). This

finding is interesting because numerous observations

suggest that inflammatory factors may underlie metabolic

diseases such as obesity. In fact, macrophages infiltrate

adipose tissue in obesity conditions (Weisberg et al. 2003;

Xu et al. 2003) and promote lymphangiogenesis in mouse

models of inflammatory disease. Accordingly, the inflammatory

process described in Prox1+/ mice may have

contributed to adipose tissue accumulation in this mouse

model (Harvey et al. 2005).

Finally, a recent study using hypercholesterolemic apolipoprotein

E (apoE)-null mice revealed that the hypercholesterolemia

in these mice is associated with tissue

swelling, lymphatic leakiness, and decreased lymphatic

transport of fluid (Lim et al. 2009). In these animals, the

mutant capillary lymphatic vessels are significantly dilated

and the collecting vessels show remarkably reduced

smooth muscle cell recruitment (Lim et al. 2009). These

data suggested that hypercholesterolemia in apoE-null

mice is associated with lymphatic vessel dysfunction. In

the next few years, researchers should strive to determine

whether some forms of lymphatic dysfunction are responsible

for human obesity syndromes.

Hypertension

A high-salt diet (HSD) is one of the causes of hypertension.

A recent work has demonstrated that, in HSDs,

lymphatic vessels play a role in blood pressure buffering

(Fig. 2; Machnik et al. 2009). This process is mediated by

mononuclear phagocyte system (MPS)-driven tonicityresponsive

enhancer-binding protein (TonEBP) and VEGFC

signaling. This signaling induces lymphatic vessel

hyperplasia, resulting in increased lymphatic drainage

capacity, and thus contributes to interstitial fluid and

blood pressure homeostasis (Machnik et al. 2009). In mice

fed with HSDs, Na+ accumulates in the interstitium of

the skin, leading to hyperplasia of lymphatic vessels: This

hyperplasia is promoted by VEGF-C that is secreted from

MPS cells (Machnik et al. 2009). Additionally, individuals

with refractory hypertension have higher concentrations

of plasma VEGF-C (Machnik et al. 2009). These findings

suggest that deregulation of TonEBP–VEGF-C signaling

may be related to hypertension in humans, and that

lymphatic vessel hyperplasia is required to regulate salt

storage in the interstitium. Alternatively, HSD-caused

hypertension might up-regulate TonEBP–VEGF-C signaling

to compensate for elevated blood pressure by promoting

lymphangiogenesis. 

Perspectives

Our knowledge of lymphatic vasculature-associated diseases has been expanded significantly in the last few years, mainly because of the identification of novel regulators of the lymphatic vasculature and the generation of valuable animal models. As this flow of new information continues, we should expect to uncover novel molecular mechanisms underlying pathologic processes that could facilitate the design of therapeutic strategies against cancer, inflammatory conditions, and metabolic diseases.

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