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Rinnakkaistallenteet Terveystieteiden tiedekunta
2017
Cardiac Lymphatics - A New Avenue for Therapeutics?
Vuorio Taina
Elsevier BV
Tieteelliset aikakauslehtiartikkelit
© Elsevier Ltd
CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.tem.2016.12.002
https://erepo.uef.fi/handle/123456789/7914
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Cardiac lymphatics, a new avenue for therapeutics?
1 2
Taina Vuorio1, Annakaisa Tirronen1, Seppo Ylä-Herttuala1,2 3
4
1Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular 5
Sciences, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland; 2 Kuopio 6
University Hospital, Science Service Center and Gene Therapy Unit, P.O. Box 1777, FIN-70211 7
Kuopio, Finland 8
http://www.uef.fi/en/web/aivi/seppo-ylaherttuala-group 9
10
Correspondence to S.Y-H. Tel. +358 403552075, seppo.ylaherttuala@uef.fi.
11 12 13
Keywords 14
atherosclerosis, cardiac lymphatic vessels, myocardial infarction, reverse cholesterol transport, 15
therapeutics 16
17 18 19 20 21 22
Abstract 23
Progress in lymphatic vessel biology and novel imaging techniques have established the importance 24
of lymphatic vasculature as part of the cardiovascular system. Lymphatic vessel network regulates 25
many physiological processes important in the heart such as fluid balance, transport of extravasated 26
proteins and trafficking of immune cells. Therefore, lymphangiogenic therapy could be beneficial in 27
the treatment of cardiovascular diseases, for example by improving reverse cholesterol transport from 28
atherosclerotic lesions or resolving edema and fibrosis after myocardial infarction. In this review, we 29
first describe recent findings in the development and function of cardiac lymphatic vessels and 30
subsequently focus on the prospects of pro- and anti-lymphangiogenic therapies in cardiovascular 31
diseases.
32 33 34
Lymphatic vessels are novel players in cardiovascular health and disease 35
36
For decades the anatomy and function of cardiac lymphatic system was analyzed with dye injection 37
techniques in large animal models [1] and the role of cardiac lymphatics in normal physiology and 38
pathological conditions remained understudied. The identification of specific lymphatic endothelial 39
cell (LEC) (see Glossary) regulators and markers, such as Prospero Homeobox 1 (PROX1) [2], 40
Vascular Endothelial Growth Factor C (VEGF-C) [3], Vascular Endothelial Growth Factor 41
Receptor 3 (VEGFR-3) [4], Podoplanin (PDPN) [5] and Lymphatic vessel endothelial hyaluronan 42
receptor 1 (LYVE1) [6] has brought several new transgenic mouse models and sophisticated confocal 43
and multiphoton imaging methods for lymphatic research [7].
44 45
The lymphatic system is required for the maintenance of body fluid balance, chylomicron transport 46
from the intestine and immune cell transport and surveillance. The lymphatic network is composed of 47
blind-ended, thin-walled lymphatic capillaries that take up extra interstitial fluid, lymphatic collector 48
vessels dedicated to the transport of the fluid, andlymph nodes that are responsible for immune cell 49
responses. LECs are loosely connected with button-like junctions that allow the entry of fluid, 50
proteins and leukocytes into the lymphatic capillaries. Lymph flows from capillaries into the valve- 51
containing lymphatic collectors and through the thoracic duct and right lymphatic trunk into venous 52
circulation [8].
53 54
As in other parts of the body, the heart has an extensive lymphatic network that regulates and 55
maintains the correct fluid balance. The obstruction of the cardiac lymph flow in the healthy heart of 56
an experimental animal can lead to severe cardiac edema, left ventricular dysfunction and 57
hemorrhages (reviewed in [1]). On the other hand, the development of new lymphatics 58
(lymphangiogenesis) may be favorable in delaying atherosclerotic plaque formation within coronary 59
arteries [9]. In addition, therapeutically induced lymphangiogenesis has shown to be beneficial during 60
the healing process after myocardial infarction (MI) as it reduces fluid retention and improves 61
inflammatory cell clearance in the cardiac tissue [10, 11]. In this review, we will introduce the 62
development and regulation of cardiac lymphatics and subsequently focus on therapeutic applications 63
for cardiovascular pathologies 64
65 66
Regulators of lymphangiogenesis 67
68
Lymphatic vessels network is formed during embryogenesis and lymphangiogenesis is also activated 69
during pathological states, such as inflammation, tumor formation and lymphedema [12]. After more 70
than a decade of debate on the origin of LECs, in 2007 Srinivasan et al. [13] confirmed that LECs 71
originate primarily from embryonic veins. They used Tie2-Cre lineage tracing, a method that utilizes 72
inducible genetical labeling of Tie2-expressing endothelial cells that can be later traced with 73
tamoxifen induction. Lymphangiogenesis starts at embryonic day 10 (E10.0) in mice [2, 14] and 74
during weeks 6-7 in human gestation when PROX1 expression is induced in a subpopulation of 75
endothelial cells in common cardinal vein, intersomitic veins and superficial venous plexus [15].
76
However, lymphatics in specific parts of the body, such as lumbar and dorsal skin regions, cannot be 77
traced to originate from Tie2 venous endothelial source [16]. In addition, mesenteric lymphatics have 78
shown to arise from a specific pool of naïve hematopoietic cells [17]. A study by Klotz et al. [10]
79
described the yolk sac haemogenic endothelium as an alternative source of cardiac LECs in mice.
80
This finding was recently challenged by Ulvmar et al. [18] proposing that Pdgfrb-Cre lineage tracing, 81
a method used by Klotz et al. labels the yolk sac haemogenic endothelium incompletely. Thus, the 82
most common origin of LECs is the embryonic veins but the exact identity of the cardiac LEC 83
progenitor remains to be determined. The function of the key factors regulating lymphangiogenesis 84
are described in the next chapters.
85 86
PROX1 87
Homeobox transcription factor PROX1 is a key molecule in the differentiation of blood and lymphatic 88
endothelial cells as it determines the specification of LECs from endothelial precursor cells during 89
embryogenesis [2]. Transcription factors SRY-Box 18 (SOX18) [19] and COUP transcription factor 2 90
(COUP-TFII) [20] are required for PROX1 activation. The expression of SOX18 is restricted to only 91
certain cells in venous endothelium and the expression is maintained in precursor LECs migrating 92
towards lymph sacs [19]. It has been speculated that SOX18 has an essential role in the early 93
induction of PROX1 but it is not sufficient for maintaining PROX1 expression later [21]. In contrast 94
to SOX18, COUP-TFII is present in all venous endothelial cells in the embryo, and it forms a 95
heterodimer with PROX1 in the cells determined for LEC specification [22]. PROX1 and VEGFR-3 96
form a positive feedback loop: VEGFR-3 gene is a direct target of PROX1 and in turn, VEGFR-3 97
signaling is essential in PROX1 expression in LECs [23].
98 99
VEGF-C and VEGFR-3 100
VEGFR-3 expression induces the budding and migration of LECs towards the mesenchyme that 101
expresses VEGF-C, the primary ligand for VEGFR-3 [24, 25]. Budding LECs coalesce and form 102
lymph sacs, which represent the first primitive lymphatic structures [14]. Both in embryos and adults, 103
VEGF-C–VEGFR-3 signaling is crucial for LEC proliferation, migration and survival [24, 25].
104
However, another VEGFR-3 ligand, VEGF-D, is not required for lymphatic vessel development 105
during embryogenesis [26]. Collagen And Calcium Binding EGF Domains 1 (CCBE1) is essential for 106
sprouting of the primitive LECs from the cardinal vein. It is highly expressed near developing 107
lymphatic vessels and particularly in the developing heart [27]. CCBE1 has been shown to enhance 108
the cleavage of pro-VEGF-C to its active form by ADAM Metallopeptidase With Thrombospondin 109
Type 1 Motif 3 (ADAMTS3) metalloproteinase [28]. In a recent study, Bui et al. [29] confirmed that a 110
complex of CCBE1-ADAMTS3 is required for proper proteolytic activation of VEGF-C but not 111
VEGF-D. It was speculated that VEGF-C is required for the growth of developing lymphatics 112
whereas VEGF-D serves as a growth factor for reactive, local lymphatic growth during inflammatory 113
reactions in adults [29]. In addition to CCBE1, neuropilin 2 (NRP2) can enhance VEGFR-3 signaling 114
by interacting with VEGFR-3 and thereby increasing the affinity of LECs towards VEGF-C [30].
115 116
Podoplanin 117
The expression of a transmembrane O-glycoprotein podoplanin (PDPN) distinguishes endothelial 118
cells inside the embryonic veins from fully budded LECs [31]. Studies in Pdpn-/- mice have suggested 119
that the expression of PDPN is required for platelet activation and aggregation by binding to a platelet 120
factor C-type lectin-like receptor 2 (CLEC-2). Platelets without PDPN function cannot aggregate and 121
therefore PDPN knockout embryos have incomplete lymphovenous separation and blood-filled 122
lymphatics [32, 33]. PDPN expression is sustained in mature LECs and it is widely used as a LEC 123
marker [5].
124 125
FOXC2 126
Forkhead Box C2 (FOXC2) is required for maturation and maintenance of collecting lymphatic 127
vessels and especially lymphatic valves [34]. In addition, FOXC2 sustains LEC quiescence and 128
stability by maintaining the intercellular junctions and cytoskeleton organization [35]. FOXC2 has 129
been shown to regulate Ras/ERK signaling pathway in LECs along with VEGFR-3. Therefore, it has 130
been speculated that FOXC2 fine-tunes the functions of VEGFR-3 during lymphangiogenesis [36].
131 132 133
Development, anatomy and function of the cardiac lymphatic system 134
135
In mice, cardiac lymphatic vessel formation starts at E11.0-12.0, a few days after the development of 136
coronary blood vessels. VEGFR-3 and PROX1 positive LECs migrate from extracardiac tissues 137
towards the outflow tract on the ventral surface of the heart. At E14.5, lymphatic vessels sprout from 138
the sinus venosus towards the ventricular surface [10]. During embryonic days E15.0-E18.0, LECs 139
migrate from the base of the heart towards the apex, following the coronary vasculature forming the 140
main precollector lymphatics [37]. In addition, cells that express macrophage marker F4-80 have 141
been shown to incorporate to lymphatic vessel walls during prenatal development [38]. After birth, 142
lymphangiogenesis continues and the lymphatic network is expanded from the subepicardium towards 143
the myocardium [37]. In mice, the lymphatic vasculature is fully developed by post-natal day 15 [10].
144
The development of cardiac lymphatic vessels is described inFigure 1.
145
In the adult heart, the cardiac lymphatic system forms a network of capillaries and precollecting 146
lymphatic vessels that are located most abundantly in the ventricles. The human left ventricular wall 147
possesses approximately 30 lymphatic vessels/mm2, significantly less than blood capillaries [39, 40].
148
There is some species-specific variation in the anatomy of cardiac lymphatics. For example, the 149
majority of lymphatics in rabbit and mouse hearts are located in the subepicardium and outer 150
myocardium, whereas in humans lymphatic capillaries can be visualized evenly in the myocardial, 151
subepicardial, and subendocardial areas (reviewed by Ratajska [40]). Cardiac lymphatic flow begins 152
from the subendocardium, runs through the myocardium and enters into the capillary lymphatic 153
plexus in the epicardium [41]. The diameter of the capillaries varies, depending on the species and 154
study, from 20 µm up to 400 µm [40]. Lymphatic capillaries converge into large valve-containing 155
collecting lymphatics that run along the left conal and left cardiac veins, and finally drain into 156
subaortic and paratracheal lymph nodes [37]. All cardiac valves also possess lymphatics, that are 157
mainly located in the basal part but also some are scattered peripherally [39].
158 159
Many congenital lymphatic disorders cause lymphatic dysfunction and lymphedema primarily in the 160
legs and feet of the individual but not in the heart, such as Milroy’s disease, associated with missense 161
mutations in VEGFR-3 tyrosine kinase domain [42] and lymphedema-distichiasis syndrome, caused 162
by mutations in the FOXC2 gene [43]. However, Noonan syndrome, affected by the upregulated 163
RAS-MAPK signaling, is associated with several signs of lymphatic dysfunction such as 164
lymphedema, lymphatic dysplasias, chylous reflux and intestinal lymphangiectasis, but also 165
congenital heart defects characterized by pulmonary valve stenosis, atrial septal defects and 166
hypertrophic cardiomyopathy [44]. Cardiac lymphatic anatomy or function has not been recorded in 167
these patients, but lymphatic dysfunction might affect the progression of heart defects as the 168
development of lymphatics is abnormal.
169 170
In addition to hereditable lymphatic dysfunctions, lymphatic vessels play a role in several heart- 171
related conditions and might have a therapeutical potential. These will be discussed in the further 172
sections of the review.
173 174 175
Lymphatic vessels in heart diseases 176
177
Heart diseases affect nearly half of the population in Western societies [45]. They are primarily 178
caused byatherosclerosis, a chronic inflammatory disease of the arteries that can cause blockage of 179
the blood flow leading to ischemia and MI. In the following sections we review the current knowledge 180
of lymphatics in the cardiac pathologies and highlight the therapeutic potential of lymphangiogenesis 181
(Table 1).
182
Atherosclerosis 183
Atherosclerosis is characterized by the formation of arterial fatty streaks and plaques loaded with 184
macrophages containing cholesterol (foam cells). A continuous recruitment of monocytes and 185
differentiation into foamy macrophages drives disease progression [46]. The so-called vulnerable 186
plaques, are especially dangerous because they can break and cause acute occlusion in large arteries in 187
the heart and other organs [47]. An attractive treatment option for atherosclerosis is to reduce 188
macrophage burden and decrease cholesterol build up within artery wall by way of a reverse 189
cholesterol transport (RCT).
190 191
RCT is a pathway responsible for cholesterol mobilization on high density lipoprotein (HDL) 192
particles from extravascular tissues such as artery wall and muscle to the liver for excretion [48]. RCT 193
is a key player in maintaining peripheral and total body cholesterol homeostasis and it could lead to 194
regression of atherosclerosis by reducing the cholesterol content within the plaques. A strong inverse 195
correlation has been established between plasma HDL cholesterol and the risk for cardiovascular 196
disease [49]. During RCT, apolipoprotein A1 containing pre- HDL removes cholesterol from 197
macrophages through the ABCA1 and ABCG1 transporters, and then travels through the bloodstream 198
to the liver for biliary excretion [48]. The exact route and mechanism for HDL RCT from peripheral 199
tissue to the circulation still remains unclear.
200 201
Recent studies suggest that lymphatic vessels could play an important role in RCT by transporting 202
HDL from interstitial tissues to the bloodstream [9, 50]. It has been shown in mice that blocking 203
lymphatic growth within the aortic wall leads to greater cholesterol retention in the aortae [9].
204
Furthermore, a surgical ablation of lymphatic vessels in the mouse skin blocked RCT without 205
impairing cholesterol efflux from macrophages indicating the importance of lymphatics in HDL 206
transport [9]. Soluble decoy VEGFR-3×Ldlr /ApoB100/100 mice and Chy×Ldlr /ApoB100/100 mice 207
both display inhibited VEGF-C – VEGFR-3 signaling thus leading to impaired lymphangiogenesis.
208
Studies with these mouse models further confirmed that lymphatic insufficiency leads to accelerated 209
atherosclerotic lesion development highlighting the importance of lymphatic vessel function in 210
cholesterol metabolism [51].
211 212
Additionally, it has been shown that restoration of lymphatic function in hypercholesterolemic mice 213
improves lipid clearance. Hypercholesterolemic ApoE mice carrying excess cholesterol in VLDL 214
and chylomicron remnant fraction displayed structural alterations in lymphatics and lower expression 215
of VEGF-C and FOXC2, which both are important factors maintaining lymphatic vasculature.
216
However, treatment of ApoE mice with a local injection of recombinant VEGF-C growth factor 217
restored lymphatic function and reduced the accumulation of cholesterol in the skin and improved 218
RCT, further emphasizing the potential of lymphangiogenic therapy as a tool for cholesterol clearance 219
[50].
220 221
Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates of LDL receptor (LDLR) by 222
binding the receptor and causing its lysosomal degradation in cells. A study employing Pcsk9 mice 223
suggests that LDL receptor (LDLR) could also play a role in lymphatic dysfunction. Atherosclerosis 224
protected Pcsk9 mice exhibited improved collecting lymphatic vessel function combined with 225
enhanced expression of LDLR on LECs whereas mice deficient of LDLR (Ldlr /hApoB100/100) were 226
shown to have defects in collecting lymphatic vessels before the atherosclerotic lesion formation in 227
LDLR-dependent manner. Treatment of Ldlr /hApoB100/100 mice with selective VEGFR-3 agonist, 228
VEGF-C152S mutant, significantly increased lymphatic vasculature in the adventitia of the aortic sinus 229
and enhanced lymphatic cellular transport. However, there is no data whether this could alter the risk 230
for atherosclerosis [52].
231 232
Most of the studies have utilized mouse models but in 2011 Kholova et al. [39] demonstrated that 233
lymphatics are also present in human coronary arteries as well as in atherosclerotic plaques.
234
Lymphatics were found in intima, media and adventitia of the arterial wall, and moreover, increased 235
medial lymphangiogenesis was present in progressive atherosclerotic lesions, which could be a natural 236
response to resolve increased inflammation and cholesterol build up [39]. Interestingly, it has been 237
shown that ApoA1 and HDL concentration in human artery wall is as high as in the plasma, thus 238
emphasizing a potential role of arterial lymphatics in carrying excess cholesterol out from 239
atherosclerotic plaques [53].
240 241
As mentioned earlier, the mechanism for HDL transport from peripheral tissue to the circulation has 242
remained unknown. Recently, it was suggested that Scavenger receptor class B member 1 (SR-BI) is 243
expressed in the lymphatic endothelium where it mediates the internalization and transport of HDL.
244
Furthermore, downregulation of SR-BI by small interfering RNAs resulted in 80% inhibition of HDL 245
uptake by LECsin vitro. It was also shown that treatment of wild type mice with an SR-BI blocking 246
antibody inhibits the transport of HDL via lymphatics by 75%. This is the first study demonstrating 247
that lymphatics can actively transport HDL cholesterol via SR-BI dependent mechanism [50].
248 249
All aforementioned studies show that functional lymphatic vasculature is responsible for cholesterol 250
removal from the artery wall and therefore, influences the atherosclerotic plaque formation.
251
Additionally, it has been proposed that HDL enters the lymphatic vasculature via SR-BI dependent 252
mechanism before reaching the bloodstream. These results suggest that therapies aimed at reducing 253
atherosclerosis by way of induced lymphangiogenesis and enhanced HDL transport may be beneficial.
254
Thus far, clinical trials aiming to increase HDL levels have failed to reduce the risk of recurrent 255
cardiovascular events [54, 55]. This accentuates that simply increasing the HDL levels is not a 256
sufficient treatment, thus targeting the mechanisms behind HDL transport could provide a better 257
therapeutic outcome.
258 259
Myocardial infarction 260
As in other parts of the body, cardiac lymphatic vessels are required for the maintenance of fluid 261
balance and immune surveillance, both important factors during and after MI. Briefly, MI is primarily 262
a manifestation of coronary artery disease where a part of the heart muscle is damaged by a blockage 263
in blood flow. In most cases, the rupture of the atherosclerotic plaques in the coronary artery wall lead 264
to a clotting cascade and complete blockage of the arterial lumen. If this ischemic condition is 265
prolonged, cardiac cells within the region die leading to the formation of an infarction scar [56].
266
Recruited inflammatory cells clear the affected area from dead cells and matrix debris and stimulate 267
regenerative processes. However, inflammatory reaction can also cause deleterious cardiac 268
remodeling [57].
269 270
Only a few studies have addressed the role of lymphatic vessels in MI. Recently, it was shown that 271
adverse remodeling of epicardial collector lymphatics in the infarcted area causes reduced lymphatic 272
flow and persistent edema [11]. Cardiac edema has been shown to induce myocardial stiffness, 273
fibrosis and LV dysfunction by increasing the amount of collagens in the ventricle walls [58]. As a 274
response to the fluid accumulation into the myocardium, lymphangiogenesis has been observed after 275
experimental LAD ligation in mice [10] and rats [11] and also in post-mortem human MI samples 276
[59].
277 278
Therapeutical lymphangiogenesis appears to be beneficial for post-MI healing. Klotz et al. showed 279
that left ventricular ejection fraction was significantly improved 14 and 21 days after MI, when 280
VEGFR-3 specific recombinant protein VEGF-C156S therapy was utilized [10]. Henri et al. [11]
281
studied the effect of microparticles carrying VEGF-CC152Sprotein, another selective VEGFR-3 ligand, 282
in an MI rat model. While, no effect was seen in infarct scar size, cardiac hypertrophy was decreased.
283
In addition, precollector remodeling was attenuated and fluid balance was improved. Both VEGF-C 284
and VEGF-D therapy has been shown to promote blood flow and healing in the rabbit and mouse hind 285
limb after ischemia, but the role of lymphangiogenesis in these studies remains questionable [60-63].
286
In addition, gene therapy trial in humans showed beneficial effects of VEGF-C therapy on angina 287
score [64, 65]. Unfortunately, lymphangiogenesis was not analyzed in these studies.
288 289
Diseases of heart valves 290
Lymphatic vessels have been shown to be present both in normal and diseased human heart valves.
291
As compared to blood vessels, lymphatic vasculature is proportionately more prominent in valves 292
than in the other areas of the heart [39]. A few publications have reported that increased 293
lymphangiogenesis is observed during valve pathologies [39, 66, 67]. Human heart valve endocarditis 294
is characterized by inflammation and abnormal growth, as well as inflammation-associated 295
lymphangiogenesis. Lymphatic vasculature within the diseased heart valves was shown to be 296
increased, both in vessel size and density [67]. However, it is not clear whether lymphangiogenesis 297
within the valves is enough to resolve the inflammation and would lymphangiogenic therapy be a 298
sufficient treatment.
299 300
Valvular stenosis involves narrowing of the valve caused by calcification and accumulation of lipids 301
and inflammatory cells [66]. Syvaranta et al. [66] showed that the lymphatic vasculature is present in 302
human stenotic aortic valves and lymphatic capillaries are associated with areas rich in inflammatory 303
cells and neovascularization. Furthermore, VEGF-D and VEGFR-3 mRNA levels in the stenotic 304
aortic valves were upregulated and conceivably are responsible for the increased lymphangiogenesis.
305
Lymphangiogenesis may provide a pathway for lipid and inflammatory cell clearance leading to 306
reduced valvular thickening.
307 308
Heart transplantation 309
The lymphangiogenesis observed after MI appears to be beneficial in resolving inflammation by 310
increasing the clearance of fluid and immune cells as well as inflammatory mediators from the injured 311
heart. However, sustained lymphangiogenesis may also provoke unwanted adverse effects by 312
supporting the transport of immune cells and antigens. Lymphatic vasculature is an important bridge 313
between the innate and adaptive immunity, and in the case of heart transplantation, it may evoke 314
adaptive immunity with serious consequences [68]. Activated antigen-presenting cells such as 315
dendritic cells migrate to the recipient´s draining lymph nodes to trigger adaptive immune responses 316
by presenting foreign antigens to T cells, thus aggravating inflammation. Because increased 317
lymphangiogenesis enables antigen-presenting cell migration, lymphangiogenesis can serve as a 318
increased exposure of lymph nodes target to modulate adverse immune reactions [68].
319 320
A recent study demonstrated that cardiac allograft ischemia-reperfusion injury in the rat heart 321
increased graft VEGF-C expression and lymphatic vessel activation. Treatment with a VEGFR-3 322
inhibitor led to reduced lymphatic vessel activation and dendritic cell maturation, subsequently 323
reducing chronic inflammation and resulting in attenuated acute and chronic rejection. Furthermore, 324
mouse studies using transplanted hearts carrying a LEC specific VEGFR-3 deletion confirmed the 325
previous results that VEGFR-3 inhibition leads to prolonged cardiac allograft survival [69].
326 327
Another method to prolong allograft heart transplant survival is to destroy the bridge between innate 328
and adaptive immunity. Azzi et al. (2016) injected microparticles containing T cell proliferation 329
inhibitors directly to the transplanted mouse heart. These microparticles mimic lymphocyte migration 330
to the lymph nodes and therefore target the activation of the adaptive immunity. Microparticles 331
delivered to draining lymph nodes successfully prolonged heart allograft survival [70].
332 333
From mice to humans?
334 335
Most of the successful cardiac studies within the lymphatic field have utilized rodent models (see 336
Table 1), which leads us to question whether the methods and results from the small animal studies 337
can be translated to complex and chronic human diseases. To resolve this question, the next important 338
step is large animal studies. There are only a few publications utilizing pig models, which demonstrate 339
that lymphedema can be treated with local injections of adenoviruses encoding lymphangiogenic 340
VEGF-C [71-73], VEGF-C156S [72] or VEGF-D [73]. The same treatment strategies could be 341
employed to treat cardiac pathologies in large animal models. There is an ongoing clinical trial for 342
enhancing cardiac angiogenesis, which emphasizes that the methods to treat MI patients are already 343
available [74]. The NOGA electroanatomical mapping and injection catheter provides a tool for 344
lymphangiogenic therapy by enabling to detect and target the borderline areas of the infarction scar 345
and to deliver the intramyocardial treatment simultaneously [74, 75]. Clearly, the tools to move on to 346
large animal studies and even to clinical trials in cardiac lymphatic field in the near future are readily 347
available.
348 349
The activation of lymphangiogenesis may result in increased exposure of lymph nodes to 350
inflammatory mediators and promote the development of metastasis (reviewed in [68]). Therefore, the 351
safety and efficacy of the lymphangiogenic therapy need to be carefully evaluated within the context 352
of each disorders and the desired therapeutical goals. Most current studies have used recombinant 353
proteins or plasmids for treatments but these generally suffer from short duration and low efficacy.
354
Another option for gene delivery would be viral vectors which efficiently carry and transduce the 355
therapeutic factor into the target cells and tissues [76]. Also, biodegradable microparticles have been 356
utilized as carriers of therapeutic factors into lymph nodes [70] and myocardial wall [11, 70]. Most 357
commonly used pro-lymphangiogenic factors are VEGFR-3 ligands VEGF-C and VEGF-D and their 358
splice variants. Other options, such as angiopoietins and fibroblast growth factors and their 359
combinations require further evaluation in this context [12, 77].
360 361
Concluding Remarks and Future Perspectives 362
363
After years of ignorance cardiac lymphatic system has become an active target for research and novel 364
findings are published at increasing pace. Recent advances in the lymphatic field have provided new 365
insights in the treatment of cardiovascular diseases (See Outstanding Questions box and Figure 2, Key 366
figure). It has been shown that the lymphatic vessels are present around atherosclerotic lesions and 367
provide an important pathway for cholesterol transportation. Therefore, novel lymphangiogenic 368
therapies could accelerate RCT leading to inhibition or regression of atherosclerosis.
369
Lymphangiogenic therapy has also been successfully utilized to resolve edema formation, 370
inflammatory cell accumulation and fibrosis during MI in mice [10, 11]. On the other hand, anti- 371
lymphangiogenic therapy could be beneficial in preventing undesired inflammation in heart 372
transplants.
373 374
In addition, modulation of lymphatic function could be beneficial in treating lipid-related diseases, 375
such as weight-gain and hypercholesterolemia. One of the first reports describing the connection 376
between lymphatics and lipids was in 2005, when Harvey et al. [78] showed that lymphatic 377
dysfunction in Prox1+/- mice caused leakage of lymph into surrounding tissues and resulted in adult- 378
onset obesity. Fat accumulation has also been observed in humans with surgical ablation of lymph 379
nodes [79] but not in other mouse models of lymphatic deficiency [8]. Evidence from the RCT studies 380
indicate that lymphatics are also required for lipoprotein transport and metabolism. It is well 381
established that lymphatics mediate chylomicron transport from intestine into bloodstream, but the 382
function of lymphatics in regulating lipid and lipoprotein uptake into the lacteals has not yet been 383
characterized. Further, the question still remains if lymphatics participate in the trafficking and 384
regulation of endogenous lipoproteins, namely VLDL and atherogenic LDL. There are many open 385
questions in the relationship of lymphatics and lipid metabolism and when clarified, new avenues 386
might open for the treatment of lipid-related diseases, primary causes of many heart pathologies.
387 388
Acknowledgements: This work was supported by Finnish Academy Center of Excellence in 389
Cardiovascular and Metabolic Diseases, Erkko Foundation and Urho Känkänen Foundation.
390 391 392
393
r: recombinant, p: plasmid, Ad: adenoviral, ip: intraperitoneal 394
395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
Table 1. Modulation of lymphangiogenesis in cardiovascular diseases
Model Species Treatment Delivery Outcome Ref
Lipoprotein
transport mouse rVEGF-C skin improved RCT [50]
Coronary artery disease
human pVEGF-2
(i.e pVEGF-C) endocardium
improved symptoms of
angina
[64]
Myocardial infarction
mouse rVEGF-C systemic (ip) improved cardiac
function [10]
rat microparticles carrying
VEGF-CC152S myocardium reduced edema,
reduced fibrosis [11]
pig AdVEGF-C myocardium prevention of MI
progression [80]
Peripheral
ischemia rabbit pVEGF-C and
rVEGF-C
angioblasty balloon and intra-arterial
improved flow [60]
Intact muscle
mouse AdVEGF-D hind limb angiogenesis,
lymphangiogenesis [61]
rabbit AdVEGF-D,
AdVEGF-C hind limb lymphangiogenesis [62]
Cardiac
allograft rat VEGF-C/D trap,
VEGFR-3 antibody intra-arterial improved allograft
survival [69]
Figure Legends 412
Figure 1. Development of cardiac lymphatic vessels 413
(A) Cardiac lymphatic endothelial cells (LECs) originate from common cardinal vein (CCV) and the 414
yolk sac hemogenic endothelium. (B) PROX1 expression is activated by COUPTF-II and SOX18 in a 415
subset of endothelial cells that are then designated for LEC specification. (C) PROX1 activates 416
VEGFR-3 expression and initiates budding of LECs from CCV. (D) LECs migrate towards the 417
systemic venous sinus along the VEGF-C gradient. NRP2 and a complex of CCBE1 and ADAMTS3 418
enhance VEGF-C signaling through VEGFR-3. (E) LECs enter the heart by E12.5 and (F) begin to 419
form the first primitive lymphatic capillaries. (G) Lymphatic network expands from base towards the 420
apex following the coronary veins. (H) By P15, cardiac lymphatic network is fully developed. Panels 421
E-H adapted from Klotz et al. [10].
422 423 424
Figure 2. Key figure. Potential lymphangiogenic therapies in cardiac diseases 425
A schematic diagram illustrating the role of lymphangiogenesis in atherosclerosis and MI. (A) 426
Atherosclerosis is characterized by lipid and inflammatory cell build up within arterial walls. (B) 427
Lymphangiogenic therapy could enhance RCT leading to greater lipid clearance from atherosclerotic 428
plaques and regression of atherosclerosis through accelerated HDL turnover. HDL removes 429
cholesterol from macrophages through ABCA1 and ABCG1 and exits via SR-BI to the adventitial 430
lymphatic vessels to the blood stream. (C) Atherosclerotic plaque rupture leads to coronary artery 431
occlusion and MI. MI is followed by adverse remodeling of epicardial collector lymphatics and 432
subsequently edema. Additionally, severe inflammation and fibrosis is developed. (D) Therapeutic 433
lymphangiogenesis after MI increases the lymph flow and resolves inflammation leading to improved 434
cardiac function and healing of MI.
435 436 437 438 439 440 441 442 443 444 445 446 447 448
Glossary box 449
Atherosclerosis: A progressive disease characterized by accumulation of cholesterol, extracellural 450
matrix, fibrosis, calcium deposits and inflammatory cells within the arterial wall.
451
Edema:The abnormal accumulation of fluid in the interstitium leading to tissue swelling and pain.
452
High density lipoprotein (HDL): A small, ApoA-rich lipoprotein responsible for reverse cholesterol 453
transport of cholesterol from extrahepatic tissues to liver.
454
Lymphangiogenesis:The formation of new lymphatic vessels from pre-existing lymphatic vessels 455
Lymphatic endothelial cells (LECs):specialized form of epithelium that lines the lymphatic vessels.
456
Lymph node: An organ linked to lymphatic vessels. It is full of immune cells that recognize foreign 457
particles and remove bacteria, viruses, toxins as well as cancer cells from the body.
458
Macrophage: A white blood cell that engulfs and digests microbes, cellular debris and any foreign 459
substances. Also acts as an antigen-presenting cell to activate immune responses.
460
Myocardial infarction (MI): Cardiac muscle damage and/or necrosis due to an occlusion of a 461
coronary artery caused by atherosclerotic plaques.
462
Reverse cholesterol transport (RCT): Movement of cholesterol from peripheral tissues back to the 463
liver via plasma lipoproteins.
464
Vascular endothelial growth factor 3 (VEGFR-3): A tyrosine kinase receptor that binds vascular 465
endothelial growth factors C and D. It is essential for the development and maintenance of lymphatic 466
vessels.
467 468 469 470
Trends Box 471
Cardiac lymphatic endothelial cells originate from multiple sources. In the heart, they form a 472
network of capillary lymphatic vessels and larger collector lymphatics that drain fluid, 473
macromolecules and inflammatory cells.
474
The role of lymphatic vessels in chylomicron metabolism is well-established and lymphatics 475
are also required for reverse cholesterol transport. Therefore, lymphatics may play even a 476
more significant role in lipid and lipoprotein metabolism than previously thought.
477
Lymphangiogenic therapy may become a useful option for the treatment of cardiovascular 478
diseases, such as atherosclerosis and myocardial ischemia. In addition, anti-lymphangiogenic 479
therapy could be utilized to resolve inflammatory reaction in cardiac allografts.
480 481 482 483
Outstanding Questions Box 484
What is the role of lymphatics in lipoprotein metabolism?
485
How could arterial wall lymphangiogenesis be enhanced? Would it increase reverse 486
cholesterol transport and could it be used as a treatment for atherosclerosis?
487
What is the most applicable delivery route and method for lymphangiogenic therapy for 488
myocardial ischemia?
489
Could attenuation of lymphatic vessel activation through VEGFR-3 inhibition therapy be 490
employed in the clinics to treat heart transplantation patients to prolong cardiac allograft 491
survival?
492
What are the side effects of lymphangiogenic therapy and how could they be avoided?
493 494495 496497 498499 500501 502503 504505 506507 508509 510511 512513 514 515516 517518 519 520521 522523 524525 526527 528529 530
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