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If chylomicrons can not get into the capillaries, how do they supply to tissues?

If chylomicrons can not get into the capillaries, how do they supply to tissues?


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The transport of chylomicrons is into the lacteals mainly because they are too big to get into the capillaries and yet they later supply triglycerides in the extra hepatic tissue by traversing in the capillary bed. This seems utterly illogical and self contradicting. So something seems to have gone wrong in this line of thought.


Lymphatic transport of high-density lipoproteins and chylomicrons

1 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA. 2 Magdalen College, Oxford University, Oxford, United Kingdom.

Address correspondence to: Gwendalyn J. Randolph, Department of Pathology and Immunology, Box 8118, Washington University School of Medicine, St. Louis, Missouri 63110, USA. Phone: 314.286.2345 Fax: 314.286.2362 E-mail: [email protected] Or to: Norman E. Miller, Magdalen College, Oxford OX1 4AU, United Kingdom. Phone: 44.20.7490.3241 Fax: 44.20.7490.3241 E-mail: [email protected]

Find articles by Randolph, G. in: JCI | PubMed | Google Scholar

1 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA. 2 Magdalen College, Oxford University, Oxford, United Kingdom.

Address correspondence to: Gwendalyn J. Randolph, Department of Pathology and Immunology, Box 8118, Washington University School of Medicine, St. Louis, Missouri 63110, USA. Phone: 314.286.2345 Fax: 314.286.2362 E-mail: [email protected] Or to: Norman E. Miller, Magdalen College, Oxford OX1 4AU, United Kingdom. Phone: 44.20.7490.3241 Fax: 44.20.7490.3241 E-mail: [email protected]

The life cycles of VLDLs and most LDLs occur within plasma. By contrast, the role of HDLs in cholesterol transport from cells requires that they readily gain access to and function within interstitial fluid. Studies of lymph derived from skin, connective tissue, and adipose tissue have demonstrated that particles as large as HDLs require transport through lymphatics to return to the bloodstream during reverse cholesterol transport. Targeting HDL for therapeutic purposes will require understanding its biology in the extravascular compartment, within the interstitium and lymph, in health and disease, and we herein review the processes that mediate the transport of HDLs and chylomicrons through the lymphatic vasculature.

HDLs constitute one of four major classes of particles that transport lipids between organs, tissues, and cells (Table 1). “Good cholesterol,” the layman’s term for HDL cholesterol, makes reference to the established role of HDL in transport of cholesterol from peripheral tissues to plasma, and from plasma to the liver, where the cholesterol is processed for packaging into bile acids for excretion. This putatively anti-atherogenic (and hence “good”) biological role of HDL in supporting excretion of excess cholesterol was first postulated by Glomset and colleagues ( 1 ) and has since been termed reverse cholesterol transport ( 2 ). Subsequently, significant evidence identified HDL as a therapeutic target to combat atherosclerosis. However, recent clinical trials designed to test whether increasing plasma HDL levels is therapeutically beneficial did not demonstrate efficacy ( 3 – 5 ), and genetic studies have refuted the prediction that higher levels of plasma HDL in humans associates with protection from cardiovascular disease ( 6 ). Harnessing the role of HDL in cholesterol clearance remains of great interest, but the field now recognizes that simple measurements of plasma HDL cholesterol do not capture its ability to support cholesterol efflux from cells, including cholesterol-loaded macrophages within atherosclerotic plaques. Indeed, the most important site from which HDLs act to promote cholesterol efflux from cells is in the extracellular matrix of tissues, not in plasma. It is plausible that some persons who display high levels of plasma HDL cholesterol have poor HDL recirculation and/or function within the interstitium of tissues, leading to poor cholesterol efflux at relevant sites. This Review considers what is known about HDL cholesterol in the interstitium and its transport through lymphatic vessels as a requisite part of its role in reverse cholesterol transport. To more comprehensively consider this topic, we also discuss chylomicron transport via lymphatics, a process that may provide insight into transport of HDL from other peripheral tissues.

Major classes of lipoproteins

The major apolipoprotein of HDL is apoA1. When the total volume of interstitial fluid is taken into account and its concentration therein considered, approximately half of all apoA1 in the body is extravascular and found within interstitial fluid of peripheral organs ( 7 ). Calculations based on lipoprotein particle numbers in plasma ( 8 ) and concentrations of apoA1 and apoB (the major protein of LDL) in plasma and peripheral lymph ( 9 ) indicate that in normal human interstitial fluid there are usually more than 50 HDL particles to one LDL particle. Nonetheless, in spite of its manifest importance in relation to lipid transport in health and disease, there is a dearth of information on HDL in interstitial fluid of peripheral tissues, owing to the difficulty of obtaining sufficient fluid under physiologic conditions.

Although subcutaneous implantation of microdialysis probes provides valuable information on movement of small molecules between plasma and interstitial fluid in humans, the molecular size cut-off of the probes is too low for recovery of lipoproteins ( 10 , 11 ). Subcutaneous wicks ( 12 ) provide insufficient material for analysis of lipoprotein composition. Greater volumes are obtained from suction blisters ( 13 ), but suction blister fluid is non-physiologic, formed by separation of the dermis from epidermis ( 14 ). The only viable approach is collection of interstitial fluid as it drains from tissues via afferent lymphatic vessels before reaching LNs (Figure 1). An underlying tenet of this approach is that interstitial fluid travels down a pressure gradient from blood to lymphatic capillaries such that the composition of lymph is a close reflection of interstitial fluid itself ( 15 ). As observed in mice, a key feature enabling movement of fluid into lymph is the structure of inter-endothelial junctions, organized so that adhesions are discontinuous, like buttons on a coat, to generate flaps between the buttons (Figure 1) that can be pulled open under tension by anchoring filaments attached to the capillary, making the vessel accessible to large macromolecules ( 16 ). Transcytosis following uptake of fluid by micropinocytosis and/or receptor-mediated endocytosis may also contribute to entry of lymph into lymphatic capillaries ( 17 ).

Organization of the lymphatic vasculature. Lymphatic capillaries form blind-ended vessels in all organs (referred to as lacteals in the intestine). These vessels converge and transition into collecting lymphatic vessels that are surrounded by muscle (red lines overlaying lymphatic vessels). Collecting vessels are interrupted by LNs. Collecting vessels and the LNs are surrounded by adipose tissue. The largest collecting vessel, the thoracic duct, drains lymph collected from all organs into the bloodstream at the subclavian vein. Insets detail the structure of lymphatic capillaries with respect to lipoprotein absorption. In tissues such as skin and lung, button-like junctions separate endothelial cells and create flaps, which allow receptor-independent entry of molecules into lymph. In intestine, large pores may form at the tips. The precise artery-capillary organization is unknown. Entry of discoidal HDL into skin lymph may be mediated by receptors through binding of SR-B1 (left inset), but more work is needed to confirm this. The right inset depicts the fenestrated transition from arterial capillary to venous blood capillary around each intestinal lacteal. Smaller nutrients not packaged in chylomicrons enter the blood vasculature that leads to the portal vein, but chylomicrons are too large. They enter the lacteal vessels by means of size exclusion, which also plays a key role in routing of HDL into lymph in other organs. Macrophages and DCs endocytose at least some molecules in intestinal villi, reducing their transit into the lacteal vessels, even if they bypass entry into the blood vasculature.

The first data on human peripheral lymph lipoproteins were obtained by Reichl and colleagues, using fluid collected from a superficial vessel in the dorsum of the foot. In studies conducted between 1973 and 1989, they showed that lymphatic lipoproteins differ from those in plasma in concentration and composition. Concentrations of apoA1 and apoB were much lower than in plasma ( 18 – 20 ), and all apoB was in LDL ( 21 ). Studies with radiolabeled lipoproteins confirmed that lymph apolipoproteins were derived from plasma ( 22 ). Measurements of cholesterol-specific radioactivity in lymph several weeks after intravenous infusion of radiolabeled cholesterol indicated that this exceeded that of plasma cholesterol ( 23 ). apoA1–containing particles had a broader size spectrum in lymph than in plasma and were enriched in cholesterol of relatively high specific radioactivity ( 19 , 24 ). These studies, reviewed elsewhere in detail ( 25 ), were consistent with the concept proposed by Glomset ( 1 ) that HDLs are the transport vehicles for cholesterol from tissues. Reichl et al. ( 26 ) subsequently found that peripheral lymph contains particles with apoA1, but no apoA2, of similar size and charge as preβ-HDLs, small lipid-poor particles that Castro and Fielding ( 27 ) had shown to be the primary acceptors of cell-derived cholesterol from cultured fibroblasts. An intervention study showed that treatment with gemfibrozil, an oral fenofibrate activator of PPARα, increased apoA1 and cholesterol in lymph but not in plasma and demonstrated the potential of the method for obtaining unique insight into extravascular cholesterol transport ( 28 ).

Miller and coworkers ( 29 ) improved the lymph collection procedure, based on an original method of Engeset et al. ( 30 ), in which lymph is collected from a larger afferent lymphatic vessel in the lower leg, allowing collection at flow rates of 0.25–2.0 ml/h for several days. The lymph in this vessel is derived from skin, adipose tissue, and connective tissue. Consistent with Reichl et al. ( 18 – 21 ), lymph was essentially devoid of VLDL. With all apoB in LDLs ( 9 , 29 ), the apoA1/apoB and sphingomyelin/phosphatidylcholine ratios were greater than in plasma. Several weeks after infusion of intravenous radiolabeled cholesterol, the specific radioactivity of lymph cholesterol exceeded that of plasma cholesterol ( 31 ). The HDLs of lymph were enriched in large apoA1-containing particles with a high content of unesterified cholesterol, phospholipids, and apo E ( 7 , 9 , 29 ). At the other end of the size spectrum, the smallest apoA1-containing particles were enriched in phospholipid. Lymph also contained discoidal HDL particles ( 29 ). Such HDLs are never seen in plasma except in subjects with familial lecithin-cholesterol acyltransferase (LCAT) deficiency. Of particular relevance to cholesterol clearance from the periphery, total cholesterol concentration in lymph HDL was about 30% greater than could be explained by the transendothelial transfer of HDL from plasma, indicating that HDLs acquire cholesterol from cells within the extravascular compartment. Preβ-HDL concentration in lymph was positively and independently related to both plasma preβ-HDL and lymph α-HDL (mature, spheroidal cholesteryl ester-rich [CE-rich] HDL) concentrations, suggesting that lymph preβ-HDL particles result not only from transport out of plasma, but also from remodeling of plasma-derived α-HDL in interstitial fluid (Figure 2). Concordant results were obtained when the same particles were quantified by chromatography ( 32 ). Subsequent in vitro incubation studies showed that lipoprotein remodeling in interstitial fluid generates preβ-HDL from α-HDL, in contrast to plasma in which there is net conversion in the reverse direction ( 7 ). That the duration of these incubations was no greater than the apparent average residence time of HDL in the extracellular matrix in humans ( 33 ) suggests that this process may be an important source of preβ-HDLs in vivo.

The intravascular/extravascular cycle of HDL remodeling that maintains reverse cholesterol transport. (i) Transfer of HDL across vascular endothelium. (ii) Production of small, lipid-poor apoA1-containing preβ-HDLs in interstitial fluid through the remodeling of spheroidal CE-rich α-HDLs. (iii) Conversion of preβ-HDLs to discoidal HDLs through uptake of unesterified cholesterol (chol) and phospholipid (PL) via the ABCA1 transporters of peripheral cells. (iv) Transport of the discs via the lymphatic system to the blood via the thoracic duct. (v) Conversion of the discs to spheroidal CE-rich α-HDLs in plasma through the action of LCAT. (vi) Transfer of CE from α-HDLs to liver cells, directly via SR-B1 receptors and indirectly via CETP and apoB-containing lipoproteins (VLDLs and LDLs) that are endocytosed by apoB100 receptors. The principal function of LCAT is to generate CEs for delivery to the liver. The net production of preβ-HDLs in interstitial fluid appears to be maintained by a high ratio of active to inactive PLTP in the presence of a near-zero cholesterol esterification rate, in contrast to a high esterification rate and lower active/inactive PLTP ratio in plasma. Black arrows represent the path of apoA1 as a component of different HDLs as they move between the intravascular and extravascular compartments. Red arrows represent the flow of cholesterol, initially as unesterified cholesterol in interstitial fluid and lymph, and then as CE in blood.

By comparing cholesterol contents of size subclasses of HDLs in lymph with those in plasma, the rate of whole body cholesterol transport via lymph was estimated to average 0.89 mmol (344 mg) per day, which was compatible with published estimates of whole body cholesterol turnover by isotope dilution analysis ( 34 ). Taken together, these studies suggest that the interstitium is a major site for generation of preβ-HDL, and implicate the lymphatic vasculature as the main transit route for movement of HDL from the interstitium to the bloodstream and liver. The concept that HDL relies on lymphatics to return to the blood during reverse cholesterol transport was demonstrated in mice, in which the lymphatic vasculature was surgically or genetically disrupted in skin, leading to marked reductions in the appearance of labeled cholesterol in plasma that originated from implanted tissue macrophages ( 35 , 36 ).

Whether the number of HDL particles in interstitial fluid affects the rate of reverse cholesterol transport was assessed by intravenous infusion of reconstituted apoA1/lecithin discs, known to produce a rapid rise in plasma HDL concentration ( 37 ), into healthy humans previously given radiolabeled cholesterol ( 31 ). During seven days of continuous lymph collection, the infusion produced sequential increases in plasma HDLs, lymph preβ-HDLs, lymph cholesterol-specific radioactivity (consistent with efflux of cholesterol from tissues), and fecal bile acid excretion. No changes occurred in lymph concentrations of enzymes that collectively remodel HDL ( 38 ), including LCAT, which esterifies free cholesterol for packaging into the central core of spherical HDLs, or phospholipid transfer protein (PLTP) and CE transfer protein (CETP), which catalyze the exchange of lipids between HDL particles or to other lipoprotein subclasses. Indeed, cholesterol esterification rate and CETP activity are both very low in lymph compared with plasma ( 7 ). On the other hand, PLTP has a higher specific activity in lymph than in plasma, owing to a greater ratio of active to inactive forms ( 7 ). High specific activity of PLTP, along with absence of cholesterol esterification, which promotes conversion of preβ-HDL to α-HDL, likely contributes to the propensity of interstitial fluid to generate preβ-HDL particles with a high activity for removing cholesterol from cells ( 7 ). In light of the need to better understand the impact of CETP inhibition on cholesterol transport in man ( 3 ), the effect of CETP activity or its inhibition on the efficiency with which HDL cycles through the interstitium is of paramount importance to investigate.

Overall, the collective evidence from these studies is consistent with the scheme in Figure 2. Passage of α-HDLs into the interstitium across endothelium may occur by transcytosis through endothelial cells, as suggested in in vitro studies ( 39 , 40 ), but ongoing work in vivo suggests a 2-pore model of ultrafiltration rather than transcytosis (our unpublished observations). When the known radii of different HDL subclasses are compared with those of the pores, the shift from small to large HDL subclasses that occurs when scavenger receptor B1 (SR-B1) and CETP are reduced would be expected to produce a major reduction in total α-HDL transport into interstitial fluid, consistent with the observation that large lipoproteins do not enter the arterial wall ( 41 ). Small, lipid-poor apoA1-containing HDLs with preβ electrophoretic mobility are generated in interstitial fluid by remodeling of spheroidal α-HDLs derived from plasma. The preβ-HDLs in interstitial fluid then interact with ABCA1 transporters on extravascular cells to acquire unesterified cholesterol and phospholipid, resulting in formation of discoidal HDL, which travel to blood via the lymphatic system along with other macromolecules that exceed the radius of TNF-α (3.24 nm) ( 42 ). HDL ranges in radius from 3.82 to 5.43 nm ( 43 , 44 ). Upon re-entering blood at the thoracic duct, discoidal HDLs act as efficient substrates for LCAT, generating spheroidal α-HDLs rich in CEs. Transfer of CEs to the liver occurs by two processes: direct uptake from α-HDLs via SR-B1 and transfer to VLDLs and LDLs via CETP. The cyclical extravascular-intravascular remodeling of HDL is critical to maintaining a flow of cholesterol from peripheral cells to the liver for re-utilization and elimination as bile acids.

Peripheral lymph differs in function and composition from intestinal lymph. The latter transports newly synthesized chylomicrons, which are at least an order of magnitude greater in radius than HDL, from the ileum to the bloodstream during the absorption of ingested fat. Together with the liver, the ileum is a significant source of newly synthesized apoA1, which appears in intestinal lymph partly as a component of nascent chylomicrons and partly in combination with phospholipid and cholesterol as discoidal nascent HDL ( 45 , 46 ). The absorptive lymphatic capillaries for lipoproteins in the intestine extend as a single lymphatic vessel in each villus, termed lacteals (Figure 1). These drain into mesenteric collecting lymphatic vessels that run through mesenteric fat and actively pump lymph under muscular and neural control ( 47 – 49 ). This lymph runs through mesenteric LNs and ultimately into the thoracic duct that drains the transported chylomicron-rich lymph into the bloodstream at the left subclavian vein. It is notable that this pattern of transport results in passage of lymph-derived lipoproteins, including the nutrients collected as “fatty meal,” through not only mesenteric LNs (prior to entering the thoracic duct), but also through the heart and vasculature of the lung and subsequently other organs, where they are degraded by lipoprotein lipase. The remnant particles derived from these lipoproteins have access to the liver, strongly contrasting with non-fatty nutrients that directly enter the portal venous circulation from intestinal villi. One consequence of this route of transit is that the lung can be exposed to lipolysaccharides and other components from intestinal microbiota with an affinity for lipoproteins that enter lymph ( 50 ). Therefore, when gut leakage of microbiota is sufficiently great as to threaten organ failure, the lung is particularly susceptible ( 50 ). It is thus important that LNs, through which all lymph runs, respond to and filter absorbed lymph to protect the host against inflammatory lipoproteins. Mice lacking the lipoprotein lipase inhibitor angiopoietin-like 4, which is expressed in mesenteric LN macrophages, are susceptible to lethal inflammation within mesenteric LNs exposed to chylomicrons bearing saturated fats ( 51 ). Secretion of apoA1 by the intestine itself may also protect the host from mesenteric inflammation, given the anti-inflammatory properties of HDL ( 52 ). It would be interesting to test whether adverse responses to gut leakage are heightened when apoA1 secretion by enterocytes is selectively lost.

Lymphatic vessels from all peripheral organs, like those from intestine, converge with the thoracic duct so that lymph delivered to the venous blood supply is a mixture of intestinal lymph and lymph draining other peripheral tissues (Figure 1). It remains uncertain how similar the mechanisms are for lipoprotein uptake between intestinal lacteals and lymphatic capillaries in other organs. We favor the concept that lipoproteins enter lymph, whether lacteals or lymphatic capillaries in other organs, by mechanisms that are nonselective with respect to molecular composition of the incoming cargo or specific receptors. This view, supported by the similarity between interstitial fluid and lymph ( 15 ), could explain how a variety of exogenous molecules, including tracer dyes, dextrans, foreign proteins, and nanoparticles, readily enter lymph. This view does not preclude the possibility that active mechanisms like macropinocytosis contribute to entry of fluid into the lymphatic vessel ( 17 ). In opposition to the concept of receptor-independent entry, Lim et al. reported that SR-B1 is required for the uptake of HDL in the skin lymphatic vasculature ( 36 ), implying that HDL could become trapped in tissues by loss of SR-B1 on lymphatic capillaries. If this finding is widely applicable, the premise that assessments of lipoproteins in afferent lymph mirror those in interstitial fluid may not hold under at least some circumstances. By contrast, the presence or absence of SR-B1 does not modulate chylomicron absorption in the intestine ( 53 ). Perhaps alternative explanations exist for the proposed role of SR-B1 in HDL transit out of the skin. For instance, the SR-B1–deficient mouse accumulates very large HDL particles in the circulation ( 54 , 55 ), such that the plasma may be unable to supply the interstitium with HDL acceptors, leading to an alternative explanation for the failure of reverse cholesterol transport of exogenously administered cholesterol. Furthermore, SR-B1 plays a pivotal role in platelet function ( 56 ). Platelets are critical mediators in development of the lymphatic vasculature through their expression of CLEC2 ( 57 – 59 ), a key C-type lectin receptor for podoplanin that is widely expressed on lymphatic endothelial cells. Thus, the skin lymphatic vasculature may be abnormal in SR-B1–deficient mice, thereby negatively affecting reverse cholesterol transport without direct receptor-mediated uptake of HDL into lymphatics. On the other hand, as evidence indicates an extrahepatic role for SR-B1 in promoting cardiovascular disease ( 60 ), the possibility that either the uptake of HDL into tissue via vascular endothelium, which may at least partially require SR-B1 ( 61 ), or its egress from tissue via lymphatics ( 36 ) might contribute to poor reverse cholesterol transport, and therefore to atherosclerosis, is plausible and intriguing.

Though more is known about uptake of chylomicrons into lacteals than about uptake of lipoproteins into peripheral lymphatics, there is a paucity of literature overall in this important area. Newborn mice deficient in pleomorphic adenoma gene–like 2 (Plag2) succumb to a wasting syndrome stemming from failed chylomicron absorption ( 62 ). In this study, PLAG2 was highly expressed by enterocytes, with at least some expression by lacteals as well. Oil red O staining indicated accumulation of lipid within enterocytes, while electron micrographs revealed that chylomicrons were released from the epithelium but could not enter the lacteal. Plag2 deficiency also impeded the uptake of cholesterol into other tissues from the plasma. There has been little follow-up to this study, and it remains unclear whether chylomicron uptake is coordinated by lacteals in a PLAG2-dependent manner or if other changes related to chylomicron secretion, composition, or size account for the outcomes observed.

With regard to particle size, it is possible to develop a working model to explain why fats packaged in chylomicrons exclusively enter the lymphatic vasculature, whereas most nutrients traverse the portal venous system for primary delivery to the liver. The blood capillary network that surrounds each lacteal is fenestrated, particularly along the venous side. These fenestrations facilitate resorption of nutrients, even as they also allow ultrafiltration of molecules from the systemic vasculature into the lamina propria. However, the fenestrae are too narrow to permit passage of even the smaller size range of chylomicrons ( 63 ). Therefore, size exclusion, much like the mechanism postulated for HDL entry into skin lymphatics ( 42 ), likely accounts for why chylomicrons are directed to the periphery through the lymphatic vasculature, rather than to liver through the portal venous vasculature. The tip of the lacteal is thought to contain large pores that, in contrast to the nearby blood vessels, are sufficient in size to allow chylomicron entry ( 64 ). That the fenestrated blood vessels lie atop the lacteal around much of its exposed surface likely contributes to ensuring that smaller molecules, including hydrophilic nutrients and antigens not packaged in chylomicrons, primarily access the blood vasculature for transport, although there is a certain probability that a portion of these small molecules bypasses the fenestrated vasculature and enters the lymph (Figure 1), consistent with experimental observations. Furthermore, the rich macrophage and DC network in intestinal villi acquires many macromolecules that enter intestinal villi through robust endocytosis ( 65 ). Their collective endocytic activity protected the lacteal from absorption of tracer antigens, whereas depletion of these cells allowed increased absorption into the lacteal, with a resulting shift in the ensuing immune response ( 65 ). The study did not investigate whether the presence of macrophages and DCs affected chylomicron absorption or absorption of other nutrients. This issue deserves attention in future research because of its important implications. First, drugs engineered to target chylomicrons for transport into the lymphatic vasculature might avoid the liver toxicity sometimes associated with higher doses of drugs that are transported through the portal venous vasculature ( 66 ). On the other hand, the fact that environmental toxins like dichlorodiphenyltrichloroethane first gain access to the systemic circulation, rather than the portal circulation, where they could be detoxified by the liver, enhances the danger they pose to human health ( 67 ). Thus, for reasons ranging from maintenance of cardiovascular and immunologic health to the avoidance of drug toxicity, a better understanding is needed about how lymphatic vessels in the intestine absorb chylomicrons and other macromolecules. We believe these studies have merit in their own right and also provide a basis for future studies on HDL entry into lymphatics in the periphery.

Successful clinical interventions to improve cardiovascular health based on targeting HDL may require that we more thoroughly explore how the HDL cycle (Figure 2) is regulated and how it may differ in various tissues and organs. The propensity of macrophages to donate cholesterol to HDL during reverse cholesterol transport differs between different anatomical compartments ( 35 ). Yet the reason for this remains unclear. Is apoA1 more enriched in interstitial fluid at different anatomical sites? Or does the relative interstitial fluid space around macrophages influence reverse cholesterol transport, such that sites of inflammation, for example atherosclerotic plaque, where macrophages are aggregated would support a more sluggish rate of reverse cholesterol transport? Does the rate of interstitial fluid flow measurably influence the rate and extent of reverse cholesterol transport? Does the efficiency of lymph transport overall affect the development or reversibility of cholesterol-driven diseases like atherosclerosis?

Answering these questions requires more research on the role of lymphatics in clearing cholesterol from artery walls where atherosclerotic plaques occur. The adventitia of large arteries is supplied with lymphatic vasculature as part of the vasa vasorum, and advanced atherosclerotic plaques promote the growth of lymphatic vessels within the intima of plaques ( 68 ). Martel et al. employed a surgical technique that suggests lymphatic vessels mediate removal of cholesterol from the artery wall ( 35 ). This work, reviewed in greater detail elsewhere ( 69 ), needs to be verified in models that do not require lymphatic remodeling as part of the experiment. Achieving this goal will likely require experimental models with larger lymphatic vessels than are observed in mice. Experimental surgical interventions in the pig do not require full aortic transplant. Remarkably, delivery of labeled cholesterol esters, either in the form of LDL or HDL, to a ligated and temporarily bypassed segment of the pig thoracic aorta revealed that HDL passes through the media and enters adventitia efficiently, leading investigators in the late 1980s to conclude that HDL was likely cleared through adventitial lymphatics ( 70 , 71 ). Labeled LDL, by contrast, penetrated only into the intimal layer, indicating specificity in trafficking through the medial wall for labeled HDL.

Nonetheless, the transport of HDL in arteries is less well studied and may differ from skin, so caution should be exercised in extrapolating from studies of peripheral lymph to the interstitial fluid of diseased arteries. Furthermore, much of the apoA1 in plaque has been rendered dysfunctional ( 72 ). On the other hand, as skin is the largest organ in the body, a substantial fraction of apoA1 is continually found there, making skin a key player in the HDL cycle regardless of how lipoproteins are transported from arteries. Indeed, hypercholesterolemic mice lacking apoA1 suffer from massive sequestration of cholesterol predominantly in skin ( 73 ). Hypercholesterolemia also impairs lymphatic transport from the skin ( 74 ), but quantifying transport from other body sites, including the artery wall, will require the development of novel assays.

Because it has proven more challenging than expected to understand the HDL cycle well enough to manipulate it therapeutically, significant attention should be focused on the half of apoA1-bearing HDL particles found within interstitia. This part of the HDL life cycle remains relatively inaccessible for study, yet transit through the interstitium is certainly as critical as the period that HDL spends in plasma. Although recent studies have recognized that simple measurements of plasma HDL cholesterol are insufficient to predict efficacy in promoting cholesterol efflux, new assays to measure HDL function still focus on HDL in the plasma ( 64 ), making it impossible to determine whether some individuals have defective trafficking or activity of HDL within the interstitium. However, a critical unanswered question is whether evaluation of HDL remodeling and passage through skin, by far the largest and most accessible tissue, would be valuable or detract from our understanding of HDL in the interstitium of the artery wall. On the other hand, focusing on mechanisms that regulate passage of HDL through any interstitium, including those that enhance passage of nascent HDL into tissues and support its ability to later enter lymph loaded with large amounts of cholesterol, would likely benefit our understanding of cholesterol uptake by HDL in all tissues, including the artery wall.

Easton et al. infused reconstituted HDL and observed clearance of apoA1 from the plasma in a biphasic manner ( 75 ), with a secondary rise in apoA1 between 24 to 48 hours, consistent with two pools of HDL ( 75 , 76 ). The second pool is likely the interstitial pool of HDL, in which HDL has a mean residence time of approximately 29 hours ( 33 ). Thus, the second rise of apoA1 may mark the return of HDL to plasma after its transit through the interstitium, much of it likely in transit through the large organ of the skin. Nanjee et al. ( 31 ) observed a similar biphasic effect on plasma preβ-HDL concentration after intravenous infusion of reconstituted HDL, compatible with delayed appearance in plasma of preβ-HDLs generated from increased remodeling of plasma-derived α-HDLs in the interstitium. Detailed assessment of this biphasic clearance is warranted. If it serves as a readout of interstitial passage of HDL, an assay more accessible than lymph cannulation may emerge to allow estimates of HDL flux through the interstitium in large cohorts of people. These assessments in turn would make it possible to determine whether such information provides valuable predictors for coronary health.

The authors are grateful for insightful discussions with Mary Sorci-Thomas (Wake Forest University, Winston-Salem, North Carolina, USA) and Nick Davidson (Washington University School of Medicine, St. Louis, Missouri, USA) prior to the preparation of this article. The preparation of this article was supported in part by NIH grants HL096539 and AI049653 and a Breakthrough Award from the Kenneth Rainin Foundation to G.J. Randolph.

Conflict of interest: Norman E. Miller is a consultant to uniQure BV, Amsterdam, Netherlands.

Reference information: J Clin Invest. 2014124(3):929–935. doi:10.1172/JCI71610.


Distribution of Lymphatic Vessels

The lymphatic system comprises a network of conduits called lymphatic vessels that carry lymph unidirectionally towards the heart.

Learning Objectives

Describe the structure of the lymphatic system and its role in the immune system and blood circulation

Key Takeaways

Key Points

  • The lymph system is not a closed system. Lymph flows in one direction toward the heart.
  • Lymph nodes are most densely distributed toward the center of the body, particularly around the neck, intestines, and armpits.
  • Lymph vessels and nodes are not found within bone or nervous system tissue.
  • Afferent lymph vessels flow into lymph nodes, while efferent lymph vessels flow out of them.
  • Lymphatic capillaries are the sites of lymph fluid collection, and are distributed throughout most tissues of the body, particularly connective tissue.

Key Terms

  • lymph: A colorless, watery, bodily fluid carried by the lymphatic system, consisting mainly of white blood cells.
  • plasma: The straw-colored/pale-yellow liquid component of blood that normally holds the blood cells of whole blood in suspension.
  • Efferent: A type of vessel that flows out of a structure, such as lymph vessels that leave the spleen or lymph nodes and arterioles that leave the kidney.

The lymphatic system is a circulatory system for lymphatic fluid, comprising a network of conduits called lymphatic vessels that carry the fluid in one direction toward the heart. Its functions include providing sites for certain immune system functions and facilitating plasma circulation in the cardiovascular system. The lymphatic system is composed of many different types of lymph vessels over a wide distribution throughout the body.

Lymph Node Distribution

Lymphatic System: The lymph nodes and lymph vessels in human beings.

Lymphatic vessels are most densely distributed near lymph nodes: bundles of lymphoid tissue that filter the lymph fluid of pathogens and abnormal molecules. Adaptive immune responses usually develop within lymphatic vessels. Large lymphatic vessels can be broadly characterized into two categories based on lymph node distribution.

  • Afferent lymphatic vessels flow into a lymph node and carry unfiltered lymph fluid.
  • Efferent lymphatic vessels flow out of a lymph node and carry filtered lymph fluid. Lymph vessels that leave the thymus or spleen (which lack afferent vessels) also fall into this category.

Lymph nodes are most densely distributed around the pharynx and neck, chest, armpits, groin, and around the intestines. Afferent and efferent lymph vessels are also most concentrated in these areas so they can filter lymph fluid close to the end of the lymphatic system, where fluid is returned into the cardiovascular system. Conversely, lymph nodes are not found in the areas of the upper central nervous system, where tissue drains into cerebrospinal fluid instead of lymph, though there are some lymph vessels in the meninges. There are few lymph nodes at the ends of the limbs. The efferent lymph vessels in the left and lower side of the body drain into the left subclavian vein through the thoracic duct, while the efferent lymph vessels of the right side of the body drain into the right subclavian vein through the right lymphatic duct.

Flow Through Lymph Vessels

The lymphatic vessels start with the collection of lymph fluid from the interstitial fluid. This fluid is mainly water from plasma that leaks into the intersitial space in the tissues due to pressure forces exerted by capillaries (hydrostatic pressure) or through osmotic forces from proteins (osmotic pressure). When the pressure for interstitial fluid in the interstitial space becomes large enough it leaks into lymph capillaries, which are the site for lymph fluid collection.

Like cardiovascular capillaries, lymph capillaries are well distributed throughout most of the body’s tissues, though they are mostly absent in bone or nervous system tissue. In comparison to cardiovascular capillaries, lymphatic capillaries are larger, distributed throughout connective tissues, and have a dead end that completely prevents backflow of lymph. That means the lymphatic system is an open system with linear flow, while the cardiovascular system is a closed system with true circular flow.

Lymph flows in one direction toward the heart. Lymph vessels become larger, with better developed smooth muscle and valves to keep lymph moving forward despite the low pressure and adventia to support the lymph vessels. As the lymph vessels become larger, their function changes from collecting fluid from the tissues to propelling fluid forward. Lymph nodes found closer to the heart filter lymph fluid before it is returned to venous circulation through one of the two lymph ducts.


The Truth about Storing and Using Body Fat

Before the prepackaged food industry, fitness centers, and weight-loss programs, our ancestors worked hard to even locate a meal. They made plans, not for losing those last ten pounds to fit into a bathing suit for vacation, but rather for finding food. Today, this is why we can go long periods without eating, whether we are sick with a vanished appetite, our physical activity level has increased, or there is simply no food available. Our bodies reserve fuel for a rainy day.

One way the body stores fat involves the body transforms carbohydrates into glycogen that is in turn stored in the muscles for energy. When the muscles reach their capacity for glycogen storage, the excess is returned to the liver, where it is converted into triacylglycerols and then stored as fat.

In a similar manner, much of the triacylglycerols the body receives from food is transported to fat storehouses within the body if not used for producing energy. The chylomicrons are responsible for shuttling the triacylglycerols to various locations such as the muscles, breasts, external layers under the skin, and internal fat layers of the abdomen, thighs, and buttocks where they are stored by the body in adipose tissue for future use. How is this accomplished? Recall that chylomicrons are large lipoproteins that contain a triacylglycerol and fatty-acid core. Capillary walls contain an enzyme called lipoprotein-lipase that dismantles the triacylglycerols in the lipoproteins into fatty acids and glycerol, thus enabling these to enter into the adipose cells. Once inside the adipose cells, the fatty acids and glycerol are reassembled into triacylglycerols and stored for later use. Muscle cells may also take up the fatty acids and use them for muscular work and generating energy. When a person&rsquos energy requirements exceed the amount of available fuel presented from a recent meal or extended physical activity has exhausted glycogen energy reserves, fat reserves are retrieved for energy utilization.

As the body calls for additional energy, the adipose tissue responds by dismantling its triacylglycerols and dispensing glycerol and fatty acids directly into the blood. Upon receipt of these substances the energy-hungry cells break them down further into tiny fragments. These fragments go through a series of chemical reactions that yield energy, carbon dioxide, and water.


LDL and HDL

As triglycerides are moved from the VLDLs in your blood into your tissues, the VLDLs are converted into low-density lipoproteins, or LDLs. The LDLs are the main cholesterol transporters in your blood, but they also contain some triglycerides. The LDLs are taken up by most of your tissues, and the triglycerides and cholesterol that they carry are used in your cells. Another type of lipoprotein called high-density lipoprotein, or HDL, can pick up some triglycerides from the other lipoproteins circulating in your blood, and the HDLs can deliver their triglycerides to your liver and other tissues for energy or storage.


How Does the Circulatory System Maintain Homeostasis?

The circulatory system maintains homeostasis by the controlled and continuous flow of blood that reaches each cell in the body. The mechanisms within the circulatory system ensure that every cell maintains a constant internal environment.

The circulation of blood is vital in maintaining homeostasis, which is the regulation of the internal conditions of the body, as described in scientist David Darling's Encyclopedia of Science. Blood carries food to cells and removes waste products.

The circulatory system comprises the heart, veins, capillaries and arteries. The system moves oxygenated blood in a continuous and controlled way from the lungs and heart so that blood reaches every cell. Blood travels through a network of vessels that include capillaries that permeate every tissue of the body. Once depleted of oxygen, the blood returns to the lungs and heart.

To maintain homeostasis, the circulatory system delivers oxygen and nutrients in the blood so that they can pass into fluids surrounding the cells. There are control mechanisms within the system to ensure that specific body areas receive a supply of blood according to their needs so that they can maintain their internal equilibrium. The circulatory system also facilitates the removal of waste products, carrying them away in plasma.


H + + HbO2 ←→ H + Hb + O2

Hemoglobin binding to oxygen is dependent on oxygen partial pressure, as depicted in the above graph. Where is oxygen partial pressure likely to be the highest?

Oxygen partial pressure is likely to be highest in the lung capillaries, as this is where oxygen will be "loaded" on to hemoglobin molecules for transportation to the tissues. Since binding affinity increases with oxygen partial pressure, one would also expect red blood cells in lung capillaries to bind the strongest to oxygen, which allows hemoglobin saturation in the lungs.

Example Question #1 : Pulmonary And Systemic Circuits

A man is diagnosed with increased pulmonary capillary resistance. As a result, which part of the heart would be expected to increase in muscle mass?

Right ventricle and left atrium

Increased pulmonary resistance means that it will be more difficult to pump blood into the lungs. The right ventricle, which performs this function, will compensate by increasing in muscle mass. The left atrium will not increase in muscle mass because it receives blood from the lungs and pumps blood into the left ventricle its muscle mass will likely be unaffected.

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Houston team one step closer to growing capillaries

Rice, Baylor College of Medicine make necessary step on road to 3-D bioprinting

HOUSTON — (July 10, 2017) — In their work toward 3-D printing transplantable tissues and organs, bioengineers and scientists from Rice University and Baylor College of Medicine have demonstrated a key step on the path to generate implantable tissues with functioning capillaries.

Researchers from Rice University and Baylor College of Medicine have shown they initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries. In microscopic images taken a different times during a weeklong experiment, researchers tracked the changes in cells (green) and cell nuclei (orange) using fluorescent markers. (Photo by Jeff Fitlow/Rice University)

In a paper published online in the journal Biomaterials Science, a team from the laboratories of Rice bioengineer Jordan Miller and Baylor College of Medicine biophysicist Mary Dickinson showed how to use a combination of human endothelial cells and mesenchymal stem cells to initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries.

The work is an important step with fragile endothelial cells (ECs) made from “induced pluripotent stem cells,” or iPSCs, a type of cell that can potentially be made from the cells of any human patient. Because iPSCs can be patient-specific, researchers hope to find ways of using them to generate tissues and replacement organs that can be transplanted without risk of rejection by a patient’s immune system. But the fragility of endothelial cells during laboratory growth has limited the utilization of this critical cell type, which is found in all vasculature.

“Our work has important therapeutic implications because we demonstrate utilization of human cells and the ability to live-monitor their tubulogenesis potential as they form primitive vessel networks,” said study lead author Gisele Calderon, a graduate student in Miller’s Physiologic Systems Engineering and Advanced Materials Laboratory.

“We’ve confirmed that these cells have the capacity to form capillary-like structures, both in a natural material called fibrin and in a semisynthetic material called gelatin methacrylate, or GelMA,” Calderon said. “The GelMA finding is particularly interesting because it is something we can readily 3-D print for future tissue-engineering applications.”

Gisele Calderon (left) and Patricia Thai. (Photo by Jeff Fitlow/Rice University)

Tissue engineering, also known as regenerative medicine, is a field aimed at integrating advances in stem cell biology and materials science to grow transplantable replacement tissues and organs. While tissue engineers have found dozens of ways to coax stems cells into forming specific kinds of cells and tissues, they still cannot grow tissues with vasculature — capillaries and the larger blood vessels that can supply the tissues with life-giving blood. Without vascularization, tissues more than a few millimeters in thickness will die due to lack of nutrients, so finding a way to grow tissues with blood vessels is one of the most sought-after advances in the field.

Miller, who earned his Ph.D. at Rice in 2008, has studied vascularization in tissue engineering for more than 14 years. During his postdoctoral studies at the University of Pennsylvania, he also became heavily involved in the open-source 3-D printing movement, and his work at Rice combines both.

“Ultimately, we’d like to 3-D print with living cells, a process known as 3-D bioprinting, to create fully vascularized tissues for therapeutic applications,” said Miller, assistant professor of bioengineering. “To get there, we have to better understand the mechanical and physiological aspects of new blood-vessel formation and the ways that bioprinting impacts those processes. We are using 3-D bioprinting to build tissues with large vessels that we can connect to pumps, and are integrating that strategy with these iPS-ECs to help us form the smallest capillaries to better nourish the new tissue.”

Each of the trillions of living cells in the human body are constantly supplied with oxygen and nutrients by tiny blood vessels known as capillaries. Measuring just a few thousandths of a millimeter in diameter, some capillaries are so narrow that individual blood cells must squeeze through them in single-file. Capillaries are made entirely from networks of endothelial cells, the type of cell that lines the inner surface of every blood vessel in the human body.

In the process of tubulogenesis — the first step to making capillaries — endothelial cells undergo a series of changes. First, they form small, empty chambers called vacuoles, and then they connect with neighboring cells, linking the vacuoles together to form endothelial-lined tubes that can eventually become capillaries.

“We expect our findings will benefit biological studies of vasculogenesis and will have applications in tissue engineering to prevascularize tissue constructs that are fabricated with advanced photo-patterning and three-dimensional printing,” said Dickinson, the Kyle and Josephine Morrow Chair in Molecular Physiology and Biophysics at Baylor College of Medicine and adjunct professor of bioengineering at Rice.

In the study, Calderon, Rice undergraduate Patricia Thai and colleagues investigated whether commercially available endothelial cells grown from iPSCs had tubulogenic potential. The test examined this potential in two types of semisolid gels — fibrin and GelMA. Finally, the researchers also investigated whether a second type of stem cell, human mesenchymal stem cells, could improve the likelihood of tubulogenesis.

Calderon said fibrin was chosen for the experiment because it’s a natural material that’s known to induce tubulogenesis for wound healing. As such, the researchers expected endothelial cells would be induced to form tubules in fibrin.

Calderon said the first step in the experiments was to develop a third-generation lentivirus reporter to genetically modify the cells to produce two types of fluorescent protein, one located only in the nucleus and another throughout the cell. This permanent genetic modification allowed the team to noninvasively observe the cell morphology and also identify the action of each individual cell for later quantitative measurements. Next, the cells were mixed with fibrin and incubated for a week. Several times per day, Calderon and Thai used microscopes to photograph the growing samples. Thanks to the two fluorescent markers, time-lapse images revealed how the cells were progressing on their tubulogenic odyssey.

Calderon conducted advanced confocal microscopy at the Optical Imaging and Vital Microscopy Core facility at Baylor College of Medicine. Calderon and Thai then used an open-source software called FARSIGHT to quantitatively analyze the 3-D growth patterns and development character of the tubulogenenic networks in each sample. In fibrin, the team found robust tubule formation, as expected. They also found that endothelial cells had a more difficult time forming viable tubules in GelMA, a mix of denatured collagen that was chemically modified with methacrylates to allow rapid photopolymerization.

Over several months and dozens of experiments the team developed a workflow to produce robust tubulogenesis in GelMA, Calderon said. This involved adding mesenchymal stem cells, another type of adult human stem cell that had previously been shown to stabilize the formation of tubules.

Miller said that while clinical applications of 3-D bioprinting are expected to advance rapidly over the next few decades, even small tissue samples with working capillary networks could find use much more quickly for laboratory applications like drug testing.

“You could foresee using these three-dimensional, printed tissues to provide a more accurate representation of how our bodies will respond to a drug,” Miller said. “Preclinical human testing of new drugs today is done with flat two-dimensional human tissue cultures. But it is well-known that cells often behave differently in three-dimensional tissues than they do in two-dimensional cultures. There’s hope that testing drugs in more realistic three-dimensional cultures will lower overall drug development costs. And the potential to build tissue constructs made from a particular patient represents the ultimate test bed for personalized medicine. We could screen dozens of potential drug cocktails on this type of generated tissue sample to identify candidates that will work best for that patient.”

Additional co-authors include Bagrat Grigoryan of Rice, Chih-Wei Hsu of Baylor College of Medicine and Sydney Gibson of both Rice and Baylor College of Medicine. The research was supported by the Gulf Coast Consortia’s John S. Dunn Collaborative Research Fund, the Cancer Prevention and Research Institute of Texas and the National Institutes of Health. Calderon, Grigoryan and Gibson were also supported by national graduate research fellowships from the National Science Foundation.


Exercise and Capillary Function

Increased capillary density allows for greater oxygen transport to your muscles, improving their ability to perform intense exercise. In addition to improving muscle function by increasing capillary density, exercise improves capillary function regardless of capillary density. Researchers at the Peninsula Medical School in Exeter, U.K., found support for this in a 2009 study. In addition to reporting that capillary function improves with exercise, they explored the decline in capillary function with age, finding that exercise helps prevent this decline.


Houston team one step closer to growing capillaries

IMAGE: Researchers from Rice University and Baylor College of Medicine have shown they can initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries. In microscopic images. view more

In their work toward 3-D printing transplantable tissues and organs, bioengineers and scientists from Rice University and Baylor College of Medicine have demonstrated a key step on the path to generate implantable tissues with functioning capillaries.

In a paper published online in the journal Biomaterials Science, a team from the laboratories of Rice bioengineer Jordan Miller and Baylor College of Medicine biophysicist Mary Dickinson showed how to use a combination of human endothelial cells and mesenchymal stem cells to initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries.

The work is an important step with fragile endothelial cells (ECs) made from "induced pluripotent stem cells," or iPSCs, a type of cell that can potentially be made from the cells of any human patient. Because iPSCs can be patient-specific, researchers hope to find ways of using them to generate tissues and replacement organs that can be transplanted without risk of rejection by a patient's immune system. But the fragility of endothelial cells during laboratory growth has limited the utilization of this critical cell type, which is found in all vasculature.

"Our work has important therapeutic implications because we demonstrate utilization of human cells and the ability to live-monitor their tubulogenesis potential as they form primitive vessel networks," said study lead author Gisele Calderon, a graduate student in Miller's Physiologic Systems Engineering and Advanced Materials Laboratory.

"We've confirmed that these cells have the capacity to form capillary-like structures, both in a natural material called fibrin and in a semisynthetic material called gelatin methacrylate, or GelMA," Calderon said. "The GelMA finding is particularly interesting because it is something we can readily 3-D print for future tissue-engineering applications."

Tissue engineering, also known as regenerative medicine, is a field aimed at integrating advances in stem cell biology and materials science to grow transplantable replacement tissues and organs. While tissue engineers have found dozens of ways to coax stems cells into forming specific kinds of cells and tissues, they still cannot grow tissues with vasculature -- capillaries and the larger blood vessels that can supply the tissues with life-giving blood. Without vascularization, tissues more than a few millimeters in thickness will die due to lack of nutrients, so finding a way to grow tissues with blood vessels is one of the most sought-after advances in the field.

Miller, who earned his Ph.D. at Rice in 2008, has studied vascularization in tissue engineering for more than 14 years. During his postdoctoral studies at the University of Pennsylvania, he also became heavily involved in the open-source 3-D printing movement, and his work at Rice combines both.

"Ultimately, we'd like to 3-D print with living cells, a process known as 3-D bioprinting, to create fully vascularized tissues for therapeutic applications," said Miller, assistant professor of bioengineering. "To get there, we have to better understand the mechanical and physiological aspects of new blood-vessel formation and the ways that bioprinting impacts those processes. We are using 3-D bioprinting to build tissues with large vessels that we can connect to pumps, and are integrating that strategy with these iPS-ECs to help us form the smallest capillaries to better nourish the new tissue."

Each of the trillions of living cells in the human body are constantly supplied with oxygen and nutrients by tiny blood vessels known as capillaries. Measuring just a few thousandths of a millimeter in diameter, some capillaries are so narrow that individual blood cells must squeeze through them in single-file. Capillaries are made entirely from networks of endothelial cells, the type of cell that lines the inner surface of every blood vessel in the human body.

In the process of tubulogenesis -- the first step to making capillaries -- endothelial cells undergo a series of changes. First, they form small, empty chambers called vacuoles, and then they connect with neighboring cells, linking the vacuoles together to form endothelial-lined tubes that can eventually become capillaries.

"We expect our findings will benefit biological studies of vasculogenesis and will have applications in tissue engineering to prevascularize tissue constructs that are fabricated with advanced photo-patterning and three-dimensional printing," said Dickinson, the Kyle and Josephine Morrow Chair in Molecular Physiology and Biophysics at Baylor College of Medicine and adjunct professor of bioengineering at Rice.

In the study, Calderon, Rice undergraduate Patricia Thai and colleagues investigated whether commercially available endothelial cells grown from iPSCs had tubulogenic potential. The test examined this potential in two types of semisolid gels -- fibrin and GelMA. Finally, the researchers also investigated whether a second type of stem cell, human mesenchymal stem cells, could improve the likelihood of tubulogenesis.

Calderon said fibrin was chosen for the experiment because it's a natural material that's known to induce tubulogenesis for wound healing. As such, the researchers expected endothelial cells would be induced to form tubules in fibrin.

Calderon said the first step in the experiments was to develop a third-generation lentivirus reporter to genetically modify the cells to produce two types of fluorescent protein, one located only in the nucleus and another throughout the cell. This permanent genetic modification allowed the team to noninvasively observe the cell morphology and also identify the action of each individual cell for later quantitative measurements. Next, the cells were mixed with fibrin and incubated for a week. Several times per day, Calderon and Thai used microscopes to photograph the growing samples. Thanks to the two fluorescent markers, time-lapse images revealed how the cells were progressing on their tubulogenic odyssey.

Calderon conducted advanced confocal microscopy at the Optical Imaging and Vital Microscopy Core facility at Baylor College of Medicine. Calderon and Thai then used an open-source software called FARSIGHT to quantitatively analyze the 3-D growth patterns and development character of the tubulogenenic networks in each sample. In fibrin, the team found robust tubule formation, as expected. They also found that endothelial cells had a more difficult time forming viable tubules in GelMA, a mix of denatured collagen that was chemically modified with methacrylates to allow rapid photopolymerization.

Over several months and dozens of experiments the team developed a workflow to produce robust tubulogenesis in GelMA, Calderon said. This involved adding mesenchymal stem cells, another type of adult human stem cell that had previously been shown to stabilize the formation of tubules.

Miller said that while clinical applications of 3-D bioprinting are expected to advance rapidly over the next few decades, even small tissue samples with working capillary networks could find use much more quickly for laboratory applications like drug testing.

"You could foresee using these three-dimensional, printed tissues to provide a more accurate representation of how our bodies will respond to a drug," Miller said. "Preclinical human testing of new drugs today is done with flat two-dimensional human tissue cultures. But it is well-known that cells often behave differently in three-dimensional tissues than they do in two-dimensional cultures. There's hope that testing drugs in more realistic three-dimensional cultures will lower overall drug development costs. And the potential to build tissue constructs made from a particular patient represents the ultimate test bed for personalized medicine. We could screen dozens of potential drug cocktails on this type of generated tissue sample to identify candidates that will work best for that patient."

Additional co-authors include Bagrat Grigoryan of Rice, Chih-Wei Hsu of Baylor College of Medicine and Sydney Gibson of both Rice and Baylor College of Medicine. The research was supported by the Gulf Coast Consortia's John S. Dunn Collaborative Research Fund, the Cancer Prevention and Research Institute of Texas and the National Institutes of Health. Calderon, Grigoryan and Gibson were also supported by national graduate research fellowships from the National Science Foundation.

The DOI of the Biomaterials Science paper is: 10.1039/C7BM00223H

Related research stories from Rice:

Modified laser cutter prints 3-D objects from powder -- Feb. 22, 2016

Open-source laser fabrication lowers costs for cancer research -- Jan. 26, 2016

Researchers create transplantation model for 3-D printed constructs -- Nov. 3, 2015

This release can be found online at news.rice.edu.

Follow Rice News and Media Relations via Twitter @RiceUNews

Located on a 300-acre forested campus in Houston, Rice University is consistently ranked among the nation's top 20 universities by U.S. News & World Report. Rice has highly respected schools of Architecture, Business, Continuing Studies, Engineering, Humanities, Music, Natural Sciences and Social Sciences and is home to the Baker Institute for Public Policy. With 3,879 undergraduates and 2,861 graduate students, Rice's undergraduate student-to-faculty ratio is 6-to-1. Its residential college system builds close-knit communities and lifelong friendships, just one reason why Rice is ranked No. 1 for happiest students and for lots of race/class interaction by the Princeton Review. Rice is also rated as a best value among private universities by Kiplinger's Personal Finance. To read "What they're saying about Rice," go to http://tinyurl. com/ RiceUniversityoverview.

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