Aged Lymphatic Contractility: Recent Answers and New Questions

Abstract

An overview is presented of recent findings related to biology of aging of the lymph transport system. The authors discuss recently obtained data on the aging-associated alterations of lymphatic contractility in thoracic duct and mesenteric lymphatic vessels; on comparisons of function of aged mesenteric lymphatic vessels in situ versus isolated specimens and important conclusions which arose from these studies; on aging-associated changes in functional status of mast cells located close to aged mesenteric lymphatic vessels; on evidence of presence of oxidative stress in aged lymphatic vessels and changes in arrangement of muscle cells in their walls. The authors conclude that future continuation of the research efforts in this area is necessary and will be able to provide not only novel fundamental knowledge on the biology of lymphatic aging, but also will create solid foundation for the subsequent developments of lymphatic-oriented therapeutic interventions in many diseases of the elderly.

The major task of the lymphatic system is lymph transport. This system of initial capillaries, transporting vessels, and nodes is designed to transport fluid, soluble molecules, and immune cells from the interstitium through the lymph nodes to the central veins. This system also provides the transportation route for inflammatory mediators, the products of tissue injury/destruction, foreign substances, and tumor cells. Dysfunctional lymph transport can result in a wide range of disturbances, including edema, altered immune cell trafficking, depressed immune function, and impaired lipid metabolism. The lack of knowledge on how aging affects lymphatic vessels certainly contributes to the situation when researchers and clinicians ignore lymph transport-related components of various diseases. Thus, investigation of the mechanisms affecting lymphatic contractile function during aging are extremely important for ongoing attempts to better understand the lymphatic system and to discover the pathogenesis and effective treatment of various aging-associated disorders.

Until recently, there were no published reports on systematic studies on aging-associated changes in the active lymph pumps. Due to the profound difficulty of measuring lymph flow in vivo, there are only a few reports demonstrating the measurements of reduced lymph flow in aged animals.^1–3 In particular, it was reported³ that aging significantly decreases lymph flow from the main mesenteric lymph duct by ∼60% between ages of 3 and 22 mo in rats.

During the last years, we obtained important functional and molecular evidence of the aging-associated alterations of contractility in lymphatic vessels, which already widened our knowledge on the biology of aged lymph flow. On the other hand, our recent studies raise new important questions that may link several diseases to impaired coordination between lymphatic function and function of other systems in the aged body. While this review provides some recent answers on current questions in biology of lymphatic aging, we also will italacize through all of the text the new important questions which have arisen on basis of these new findings, thus illustrating therefore the widening of our knowledge of lymphatic research and studies in lymphatic biology.

Recently we performed experiments with analysis of the contractile activity of the isolated aged rat thoracic duct segments and compared these data with those obtained from their adult counterparts.⁴ We found various signs of the age-related alterations of active pump in the rat thoracic duct (TD). The transmural pressure/stretch-dependent modulation of lymphatic contractility is one of the principle regulatory mechanisms of lymphatic pumping that serves a goal to adapt lymphatic transport to the different lymphatic preloads.^5–10 The alterations in stretch-related regulatory mechanisms in 24-mo old segments indicate that both pacemaking and contractile machinery are involved in age-related changes of active lymph pump in rat TD. Lowered lymphatic tone in aged TD segments, together with decreased lymphatic contraction amplitude, may be considered an indicator of age-related weakening of muscle cells and their diminished ability to create enough force to maintain the level of tone and contractile force appropriate to the lymphatic preloads. At a comparatively low pressure level of 1 cm H₂O, the contraction amplitude is moderately lower in aged segments of TD. At higher pressure levels, the contraction amplitude is diminished in a greater degree in aged TD and reaches the statistically significant different levels between 9-mo and 24-mo old specimens. This negative age-related inotropy in thoracic duct was accompanied with alteration in function of lymphatic pacemaking: frequency of lymphatic contraction was diminished, especially at high levels of transmural pressure (5 cm H₂O). Such negative age-related chronotropy in TD, together with negative inotropy, led to a greatly decreased functional pump flow, indicating the diminished pumping ability of aged TD. The differences between stretch-induced responses in adult and aged animals are greater at higher levels of transmural pressure, suggesting also a diminished ability of aged TD to adapt its contractility to increased preloads. Thus, aging of the TD leads to decreases in its functional reserves to adapt the contractility, and pumping to the increased levels of lymph inflow in it. At higher levels of preload in the aged TD, its possibility to serve the increased demand in pumping through the duct will be diminished. Consequently, partial or complete failure to provide the adequate transport of lymph through the duct may occur.

Aging also alters the contraction-initiated self-regulatory mechanism in TD that serves to maintain lymphatic contractions in efficient energy-saved mode.¹¹ Even though administration of N-Nitro-L-arginine methyl ester hydrochloride (L-NAME) application at 100 μM in aged thoracic duct caused increases of lymphatic tone to the same values observed in adult specimens, the contraction amplitude and contraction frequency did not demonstrate the regular patterns of changes after NO-synthase blockade, as we observed for 9-mo segments in F-344 rats or as we have previously shown in TD segments obtained from adult Sprague-Dawley rats.¹¹ Such alterations in the contraction-initiated self-maintained regulation in TD may also have an impact on its pumping ability and be responsible for weakening of its contractile reserves. The fact that fractional pump flow (pumping) was slightly increased in aged thoracic duct in the presence of L-NAME may indicate the presence of inhibitory influences of the constant release of nitric oxide in the aged thoracic duct independent from the phasic contractions.

The findings described above correlate well with the data obtained in experiments with increased imposed flow in thoracic duct. In these experiments, the 9-mo old TD segments demonstrated the regular^9,10,12,13 pattern of imposed-flow induced inhibition. Administration of L-NAME completely abolished this inhibition. Fractional pump flow in 9-mo old specimens remained unchanged, even at high levels of imposed flow after NO-synthase blockade. Several studies have shown the involvement of the lymphatic endothelium in the NO-mediated modulation of lymphatic contractile activity.^11,14–16 In particular, specific immunostaining of endothelial nitric oxide synthase (eNOS) was demonstrated in endothelial cells of bovine lymphatics¹⁷ and in rat lymphatic endothelial cells.¹⁸ In functional experiments it was demonstrated that precontracted dog thoracic duct rings relaxed to acetylcholine (ACh) or sodium nitroprusside (SNP).¹⁹ Removal of the endothelium blocked the ACh-induced relaxations, which were suppressed or abolished by pre-treatment with oxyhemoglobin, methylene blue, and L-NMMA. In other studies, ACh produced negative chronotropic and inotropic effects on the spontaneous contractions in isolated bovine mesenteric lymphatics²⁰ that were dependent on the endothelium. Furthermore L-NMMA suppressed these responses, but did not eliminate them. In addition, it had been demonstrated that L-arginine completely reversed the inhibition of the ACh-induced responses by L-NMMA. The sources of NO involved in the regulation of lymphatic pump activity of rat mesentery were studied in situ.²¹ L-NAME (nonspecific NOS-blocker) caused increases in contraction frequency and pump flow in lymphatics, and decreases in the lymphatic diameters. L-Arginine reversed these effects while aminoquanidine, a iNOS inhibitor, had no effect. The authors conclude that endogenous NO inhibited lymphatic pump activity and that these effects were mediated by eNOS. Our recent studies demonstrated the involvement of NO on imposed flow induced inhibition in rat mesenteric lymphatic vessels.¹² Taken together, all literature data described above and our data obtained in this study,⁴ we concluded that in 9-mo old segments of rat TD the imposed flow-induced inhibition of the active lymph pump was completely dependent on the NO released due to the activity of eNOS.

Interestingly, TD segments from 24-mo old rats behaved differently during the increases in imposed flow. The TD segments from aged group did not exhibit any significant imposed-flow dependent inhibition of the parameters of the active lymph pump, which remained unchanged during the imposed flow elevations. We concluded that eNOS-mediated imposed flow-dependent regulatory mechanism was completely depleted in 24-mo-old TD segments. Therefore the ability of aged TD to adapt its pumping to the different levels of extrinsic lymph flow was severely altered. Moreover, the administration of L-NAME moderately increased lymphatic pumping in 24-mo-old TD segments independent from the value of imposed flow. These findings indicated the presence of a flow-independent but NO-dependent inhibition of lymphatic contractions in aged TD. We propose that this inhibition exists in aged lymphatic segments due to the activation of iNOS. To confirm the functional data on pressure- and flow-independent NO-dependent inhibition of the active lymph pump in aged TD, we performed Western blot analyses of eNOS and iNOS in samples isolated from 9-mo and 24-mo-old TD. Data obtained in these experiments clearly indicated that the relative levels of eNOS were decreased in the 24-mo old thoracic duct when compared with that of 9-mo old thoracic duct, whereas iNOS levels were dramatically increased in 24-mo old thoracic duct. The detailed functional tests of aged TD described above clearly demonstrated that it is too simplistic to attribute the diminished contractility only to the sclerosis of aged TD²² and/or to atrophy of muscle cells in its wall.^22,23

In the next study, we evaluated the aging-associated changes in pumping of mesenteric lymphatic vessels (MLV) in adult (9-mo old) and aged (24-mo old) Fisher-344 rats.²⁴ These data demonstrated a severe weakening of the lymphatic pump in aged MLV, including diminished lymphatic contraction amplitude, contraction frequency and, as a result, lymphatic pump activity. The data also suggest that the imposed flow gradient-generated shear-dependent relaxation does not exist in aged rat MLV, and the sensitivity of both adult and aged MLV to such shear cannot be eliminated by nitric oxide synthases blockade. These data provide new evidence of lymphatic regional heterogeneity for both adult and aged MLV. In MLV, a constant interplay between the tonic and phasic components of the myogenic response and the shear-dependent release of nitric oxide predominantly determines the level of contractile activity; the existence of another shear-dependent but NO-independent regulatory mechanism is likely present. Aging remarkably weakens MLV contractility, which would predispose this lymphatic network to lower total lymph flow in resting conditions and limit its ability to respond to an edemagenic challenge in the elderly.

In the discussed study,²⁴ we completed the first comprehensive evaluation of aging-associated alterations in the contractile responses of mesenteric lymphatic vessels. Our new data allow us to make several important conclusions that widen our basic knowledge of the regulatory mechanisms of mesenteric lymph flow in adulthood and senility, demonstrating therefore the complex nature of the lymphatic system. Our previous studies demonstrated regional heterogeneity in the contractile behavior of lymphatic vessels.^10,13,25 Our current studies provided the first evidence of regionally different influence of aging on different lymphatic networks. To illustrate such conclusions, we compared isolated MLV data with published data⁴ obtained from experiments utilizing isolated rat TD segments from similar groups of adult (9-mo old) and aged (24-mo old) F-344 rats and treated with similar experimental conditions.

Thus, we performed a series of tests to determine the contractile responses of MLV isolated from 9-mo-old (adult) animals to increases in wall stretch generated by different levels of transmural pressure. These tests provided adult control data for comparison to data obtained with aged MLV, and expanded our knowledge on the basic contractile characteristics of the adult mesenteric lymphatic vessels, highlighting their important differences with the lymphatic contractions in the thoracic duct. In the first set of experiments, we blocked the NOS in conditions without steady flow through the vessel segment, induced by the imposed flow. In this set of experiments, the endothelial cells in the MLV therefore only experienced shear stress due to the flow generated by the spontaneous phasic contractions of the lymphatic muscle cells. No exogenous pressure gradient favorable to flow was present in the lymphatic specimen. This contractions-generated “active” flow in MLV is responsible for phasic fluctuations in wall shear stress²⁶ and leads to the appearance of spikes of NO release by the lymphatic endothelium that are synchronous with lymphatic contractions.²⁷ By analyzing the physiological importance of such phasic contractions-generated shear-dependent regulation in thoracic duct,¹¹ we previously made following important conclusions. This large lymphatic duct, with lower resistance to flow, does not need strong, long-distance lymph active pumping and presumably because this, and due to the low variability in lymph flow patterns during the different periods of day, the thoracic duct has a somewhat simple shear-dependent regulatory mechanism to support an energy-sufficient contractile pattern solely through NO-dependent self-regulation.

In the current study²⁴ of adult mesenteric lymphatic vessels, we found that opposite to thoracic duct, the NOS blockade by L-NAME cannot induce negative inotropy. The amplitude of lymphatic contractions remained the same after NOS blockade in adult MLV even though their contraction frequency increased over all selected levels of transmural pressure under such conditions. As a result, the minute productivity of the adult MLV increased after NOS blockade. It seems unlikely that, in natural in situ conditions, the NO molecules will serve a role to limit the productivity of the active lymph pump, and thus, the increase in FPF in isolated adult mesenteric vessels after L-NAME administration looks somewhat artificial. On the other hand, the fact that NOS blockade induced a significant increase in MLV tone and frequency (therefore limiting diastolic time and diastolic filling) allows us to expect by default that the contraction amplitude in the absence of NO should go down as it does in TD at the same conditions.¹¹ Therefore, our data support the assumption that the phasic shear-dependent regulation of MLV contraction interplays with additional important mechanism(s) to control the lymphatic contractile cycle in these vessels seems to be more complex compared to TD.

Our current data, and data previously by us and others, allow us to conclude that there is a complex interplay between the influences of phasic and tonic components of the stretch-dependent myogenic responses and the influences of phasic contractions-generated NO release in MLV. In the mesenteric lymphatic network, the lymph formation and lymph flow may increase very fast with digestion. During such periods, the intralymphatic pressure may rise dramatically fast, resulting in the development of myogenic constriction,²⁸ the tonic component of which, for example, prolonged vessel constriction, by itself may lead to a local increase in vessel resistance to flow. The latter is unnecessary for lymphatic vessels during periods of high lymph formation. Stronger phasic contractions during the myogenic response (“compensatory increase in amplitude”²⁸) will support stronger fluid propulsion, higher fluctuations in wall shear, and therefore higher phasic release of NO. The latter will enhance the diastolic relaxation effect through easing the lymphangion filling and diminishing subsequently local resistance to flow. In other words, after pressure elevation, the positive phasic inotropy during the development of the myogenic phasic response would counterbalance the release of additional NO. In the rat thoracic duct, where there is lower total resistance to flow, lesser necessity for strong active pumping, and generally smaller tone-induced changes in lymphatic diameter relative to maximal diameter, the tonic and phasic components of the myogenic response may play a less functional role. Conversely, the data indicate a dominant functional importance for nitric oxide release in the control of the contractility in this largest lymphatic vessel of the body.^10,11,15,29

In our experiments with L-NAME, performed in the discussed study,²⁴ we artificially misbalanced the cascade of contractile regulatory reactions by eliminating the phasic contraction-generated shear-dependent NO release. At the same time, we did not eliminate the phasic contraction-generated shear itself so the potential existence of an additional as of yet unidentified shear-dependent, but NO-independent, mechanism for the regulation of lymphatic contractile strength in the MLV cannot be excluded. Further support of this idea can be seen in results of our experiments with NOS blockade in MLV during periods of increased imposed flow. Opposite to adult thoracic duct, L-NAME cannot completely abolish the influences of the imposed flow gradient on the MLV contractile pump, which are similar to effects of LNMMA observed by us earlier in these lymphatic vessels.¹² In conclusion, our findings in adult MLV under control and L-NAME-treated conditions provide new support for the complexity and regional variability in the mechanisms controlling lymph flow in the body, which still require additional investigation.

The objectives of the next study performed in our laboratory³⁰ were to evaluate the aging-associated changes, contractile characteristics of mesenteric lymphatic vessels (MLV), and lymph flow in vivo in 9-mo and 24-mo old Fischer-344 rats. Lymphatic diameter, contraction amplitude, contraction frequency and fractional pump flow, lymph flow velocity, wall shear stress, and minute active wall shear stress load were determined in MLV in vivo before and after L-NAME application at 100 μM. The active pumping of the aged rat mesenteric lymphatic vessels in vivo was found to be severely depleted, predominantly through the aging-associated decrease in lymphatic contractile frequency. Such changes correlate with enlargement of aged MLV, which experienced much lower minute active shear stress load than adult vessels. At the same time, pumping in aged MLV in vivo may be rapidly increased back to levels of adult vessels, predominantly through the increase in contraction frequency induced by NO elimination. Findings support the idea that in aged tissues surrounding the aged MLV, the additional source of some yet unlinked lymphatic contractions-stimulatory metabolites is counterbalanced or blocked by NO release. The comparative analysis of the control data obtained from experiments with both adult and aged MLV in vivo and from isolated vessel-based studies clearly demonstrates that ex vivo isolated lymphatic vessels exhibit identical contractile characteristics to lymphatic vessels in vivo.

With more details, in this study³⁰ we performed, for the first time, a detailed evaluation of the contractile activity of MLV and lymph flow in vivo in 9- and 24-mo-old F-344 rats. In the first part of this study, we investigated, in detail, the contractile characteristics of MLV and lymph flow in adult (9-mo) and aged animals (24-mo) under control conditions. We found that in aged animals the lymphatic vessels diameters from the same location (group III by Benoit³¹) are significantly larger than in adult animals. The end-diastolic diameters and end-systolic diameters in 24-mo old MLV were 71% and 79% greater than in their adult counterparts. At the same time, we observed only minor, nonsignificant lowering of the contraction amplitude in aged MLV versus adult vessels under control conditions (20% and 25% of diameter changes during the contractions, respectively). The aging-associated negative chronotropy was observed in all aged MLV: we noted 3-fold decrease in their contraction frequency compared to adult MLV. As a result of the described aging-associated changes in lymphatic contractile force (contraction amplitude) and pacemaking (contraction frequency), the minute active lymphatic pumping was significantly lower in aged animals. Both indices of the lymphatic pumping, AFP and FPF, were significantly diminished in the aged group with AFP 76% lower (∼4.2-fold lower) and FPF – 77% lower (∼4.3-fold lower) than the adult group.

In addition, we analyzed the aging-associated differences in the characteristics of lymph flow in rat mesentery using selected single contraction cycles of “diastole-systole” with suitable contractile cycles during each experimental condition for the 9-mo-old animals, and for the 24-mo-old animals. While the diastolic lymph flow velocity was slightly, but not significantly, higher in aged MLV, the maximal systolic lymph flow velocity was significantly (43%) lower in aged animals. Correspondingly, we did not find any aging-associated changes in calculated diastolic (resting) wall shear stress, but during the phasic contractions the lymphatic endothelial cells in aged MLV experienced a ∼3-fold reduction of the maximal systolic wall shear stress.

We also compared the rate of change of the phasic contractions-generated (i.e., active) wall shear stress in both adult and aged groups and found a dramatic ∼6.2-fold aging-associated decrease in 24-mo MLV in comparison to the 9-mo group. The phasic contractions-generated (active) minute wall shear stress “load” that the lymphatic endothelial cells experienced a minute, the active shear-frequency product (ASFP), was ∼9.7-fold lower in aged MLV.

Because of the importance of the NO molecule released by lymphatic endothelium for the regulation of lymphatic contractility and flow in adult^{11,14,16,27,32–34} and aged^4,24,29 lymphatic vessels, in the discussed study³⁰ we implemented in vivo the local NOS blockade induced by topical administration of 100 μM of L-NAME. We compared the contractile behavior of MLV and lymph flow in adult and aged groups before and after the L-NAME administration. We found that the NOS blockade with a duration of 15 min induced slight, but not significant, constriction in both adult and aged MLV. Only end-systolic diameter in aged MLV was significantly decreased by 25% after 15 min of the L-NAME application. During these small changes in lymphatic diameters, after the NOS blockade in MLV of both aged groups, the difference between the contraction amplitude in adult and aged MLV was reversed by L-NAME from slightly negative (20% lower in aged group) to positive (50% higher in aged group). The greatest observed influence of the NOS blockade was its chronotropic effect. While in adult MLV the contraction frequency was significantly increased (∼2.1-fold) 15 min after the L-NAME application, in the aged MLV this increase in the lymphatic contraction frequency was over 3.5-fold. The average contraction frequency of the aged MLV treated even only 5 min by L-NAME was 25% (although not statistically significant) higher than the contraction frequency in adult lymphatic vessels under control conditions. The main paradox of the influence of the NOS blockade in aged MLV was found when analyzing the indices of their minute productivity, AFP and FPF, which increased in both age groups as consequence of chronotropic and inotropic influences of the L-NAME administration. In adult vessels, L-NAME application was able to increase significantly both of these indices by 57% and 59%, respectively. In aged MLV, the influence of the NOS blockade was remarkably greater. Specifically, AFP was increased by 538%, and FPF by 511% compared to the control conditions after 15 min of the L-NAME administration. Such influence of the elimination of NO on aged MLV was able to not only compensate for the observed ∼4.2-fold aging-associated depletion in minute productivity in the aged MLV lymph pump in vivo but, after only 5 minutes of the L-NAME application, was able to maintain this productivity at the same level as the L-NAME-treated adult lymphatic vessels.

Another important finding from the discussed study³⁰ was that the diastolic lymph flow velocity remained unchanged during the L-NAME application in both selected aged groups. At the same time, we observed a moderate, nonsignificant, increase in the maximal systolic lymph flow velocity in the adult group, in aged MLV the maximal systolic lymph flow velocity was more than tripled after 15 min of the L-NAME treatment (∼3.6-fold increase), thus being higher than in adult MLV under the same experimental conditions. Correspondingly, we did not find any aging-associated changes in calculated diastolic (resting) wall shear stress after the NOS blockade in both selected age groups, while the maximal systolic wall shear stress was significantly increased in MLV of both selected ages (139% increase in adult and 452% increase in aged MLV). Although the absolute difference (roughly 7 dynes/cm²) is similar, the percentage as a reflection of the basal control level of the maximal systolic wall shear stress does matter. By underlying the degree of changes, we are underlying here the general tendency of lymphatic pumping in aged MLV to be increased dramatically after NO elimination towards values observed in adult animals at the same conditions. Additionally, we compared effects of the L-NAME treatment on the rate of change of the phasic contractions-generated (i.e., active) wall shear stress in both adult and aged groups. We found its dramatic ∼4.8-fold increase in 24-mo L-NAME-treated MLV as compared to aged MLV under control conditions. Such changes after L-NAME administration nearly matched the rate of change in active wall shear stress for the NOS-blocked aged MLV versus the NOS-blocked adult vessels, for which the rate did not change significantly from control during the L-NAME treatment. As a reflection of these changes the phasic contractions-generated (active) minute wall shear stress “load”, which the aged lymphatic endothelial cells experienced a minute after NOS blockade, showed the ASFP to increase by ∼25.3-fold in comparison to aged MLV in control conditions. However, the ASFP did not change significantly over the NOS-blockade in adult MLV during the same period of time.

Furthermore, in this study³⁰ we performed comparative data analysis of the contractile parameters of rat MLV obtained both in vivo and in isolated vessels experiments. We found that in control groups of 9-mo and 24-mo-old MLV all three investigated contractile characteristics, namely, contraction amplitude, contraction frequency, and fractional pump flow, obtained in isolated vessel-based experiments were not significantly different from those obtained in vivo. Moreover, after L-NAME administration, as described in this study for the in vivo experiments and previously²⁴ for isolated vessels, all three parameters of contractile activity of the 9-mo-old MLV were not significantly different between in vivo and isolated vessels data groups with the same degrees and directions of changes both in vivo and under isolated vessel conditions. The primary intriguing difference we found was in contractile chronotropy of the aged MLV in vivo after L-NAME administration as compared to the isolated vessel experiments with the same aged MLV under the same experimental conditions (NOS blockade). The contraction frequency of the 24-mo-old MLV situated in vivo increased ∼3.5-fold in comparison to the frequency of the same vessels under control conditions. As a result of this positive L-NAME-induced chronotropy and the 50% (but nonsignificant) increase of the contraction amplitude, the minute pumping (FPF) of the L-NAME-treated aged MLV was ∼6.1-fold higher than in the same aged vessels under control conditions. In other words, the NOS blockade of aged MLV in vivo was able to not only compensate the aging-associated deficiency of minute pumping in aged vessels under control conditions, but additionally to enhance their minute pumping all the way up to the levels of NOS-treated adult 9-mo MLV. In isolated aged MLV we observed much weaker, nonsignificant, positive chronotropy and a much smaller increase in pumping after L-NAME administration.²⁴

Therefore we performed, for the first time, a detailed evaluation of the parameters of the contractility of aged mesenteric lymphatic vessels in vivo and characterized lymph flow in the aged mesenteric lymphatic network.³⁰ Results were compared to the same characteristics of lymphatic contractility and flow in adult mesenteric lymphatic vessels. We performed these evaluations under control conditions and after a nitric oxide synthases blockade of 100 μM L-NAME. This allowed us to make important conclusions about the comparative roles of the NO-dependent regulatory mechanisms, which control the lymphatic contractility and lymph flow in vivo in the adult and aged body. Importantly, we performed, for the first time, the direct detailed comparison of the characteristics of the lymphatic contractility in vivo and contractile characteristics of the isolated lymphatic vessels exteriorized from the same location for both adult and aged MLV under both control conditions and after NOS synthases blockade.

On the other hand, a careful analytical comparison of the influences of the NOS blockade in the aged MLV in vivo and in isolated vessel-based studies allowed us to determine the important differences (discussed below) between functioning of the lymphatic vessels per se, and functioning of the same vessels under the additional influence of the aged tissue microenvironment.

As we mentioned above, there is little information available in literature dedicated to the aging-associated changes of lymphatic contractility and lymph flow. Therefore, the detailed evaluation of the status of lymphatic contractility and lymph flow in the aged body is an important initial task, which is able to provide additional, still mainly ignored knowledge on unknown but essential, elements of pathogenesis of many chronic disorders, which manifest or worsen with aging. While such evaluations of the aging of lymphatic functions are still in their infancy, we believe that the immediate discovery of aging-associated alterations of the regulatory mechanisms controlling lymph flow in the aged body is not possible before the completion of a careful descriptive characterization of the aging-altered parameters of lymphatic contractility and lymph flow started by this and similar studies.

The initial finding, which attracted our attention while comparing adult and aged MLV in vivo, was the fact that the aged lymphatic vessels from the same anatomical location within the mesenteric lymphatic network in rats of the same body weight have significantly greater resting lymphatic diameter. We consider this enlargement of the aged mesenteric lymphatic vessels as an indicator of the deep aging-associated remodeling of the lymphatic wall. With sparse literature data on sclerosis, enlargement, aneurysm-like formations, muscle cell atrophy, and muscle layers' disorganization in aged lymphatic vessels,^16,22,23 our current findings³⁰ create a solid foundation for detailed follow-up studies on aging-associated alterations of the biomechanical properties of lymphatic wall.

Furthermore, we found a severe negative chronotropic effect of aging on the contractility of MLV in vivo. Specifically, their contraction frequency was depleted while contraction amplitude of the aged vessels was only slightly diminished. Consequently, we observed a profound aging-associated decrease in the minute productivity of the aged MLV in vivo that we mainly linked not to the altered degree of contractile displacement of the lymphatic wall, but to the diminished number of the contractile events in aged lymphatic vessels. At the same time, we believe that we discovered additional signs of sluggishness of the contractile events in aged MLV. Substantial reduction in the rate of change in development of phasic contraction-generated wall shear stress indicates the slower development of systolic contractile force in aged lymphatic wall with slower generation of the lymph-propelling local axial pressure gradient. Greatly diminished maximal systolic lymph flow velocity may also be considered as an additional sign of slower and weaker generation of the pressure wave inside aged lymphatic vessels. This aging-associated decrease of the phasic contraction-generated (active) wall shear stress correlates with profound depletion in contraction frequency of the aged MLV. These two overlapping events of lymphatic aging cumulatively induced dramatic depletion in minute active wall shear stress load (∼9.7-fold decrease in ASFP) to lymphatic endothelial cells, which is the first time has been described for aged mesenteric lymphatic vessels. This aging-associated decrease in minute active wall shear stress load therefore creates a ground for disruption of the phasic contractions-generated shear/NO-dependent regulatory mechanisms of the lymphatic contractile events^{11,16,24,27,29} in aged mesenteric lymphatic network. Currently, there are not enough data to determine the nature of the aging-associated negative chronotropy in the MLV, existence of which was confirmed both in vivo as well as in isolated vessel-based experiments. We propose that the negative chronotropy and the consequent depletion of the minute active wall shear stress load may occur due to the yet unknown aging-associated alterations in lymphatic pacemaking. At the same time this decrease of minute active wall shear stress load will induce a reduction in the amount of phasically-generated NO through depletion of already well-described mechanisms.^11,27 Further, the diminished phasic NO release in aged MLV will consequently intensify the slowing of the lymphatic contractile events by reducing the rate of lymphatic diastolic relaxation and diastolic lymphatic filling,¹¹ and therefore will slow the aged lymphatic contractility even more. We observed this in our current studies. However, to date the complex detailed evaluation of the functional importance for this newly discovered phenomenon of the aging-associated reduction in the minute active wall shear stress load in aged MLV remains to be performed.

Next, we implemented³⁰ topical application of 100 μM of the nonselective NO synthases inhibitor, L-NAME, in order to eliminate phasic contractions-generated and steady flow-generated release of the NO in both adult and aged MLV. At low levels of lymph flow (fasted rats), we found that the topical L-NAME administration, without increase in diastolic lymph flow, was able to induce changes in lymphatic tone, chronotropy, and minute pumping of 9-mo-old MLV. However, constriction and increase of the contraction frequency during NO absence did not lead to decrease of the contraction amplitude as was found at low levels of flow for solely NO-modulated shear-dependent regulatory mechanisms of the thoracic duct contractility.^4,11 Potentially, in the MLV another mechanism prevents NOS-blockade negative inotropy by maintaining contraction amplitude at the same level. This observation supports our previously expressed hypothesis on the potential existence of an additional yet unidentified shear-dependent, but NO-independent, mechanism for the regulation of lymphatic contractile strength in the MLV.²⁴

An intriguing consequence of the topical L-NAME administration in aged MLV in vivo is the profound differences depicted in the chronotropic response between in vivo and isolated aged vessels. The contraction frequency of in vivo L-NAME-treated MLV was significantly greater than that during similar treatment in isolated vessels at transmural pressure 1 cm H₂O. Cumulatively, the minute productivity of the aged MLV in vivo was significantly greater than that in isolated vessels at transmural pressures 1 and 3 cm H₂O during the NO synthases blockade. At the same time, the L-NAME application in aged MLV in vivo was able not only to compensate the aging-associated deficit in minute pumping, but also to increase pumping in aged vessels up to levels of pumping in adult MLV. We conclude that MLV in elderly possesses considerable contractile reserves such that even “chronically developed” alterations in muscle cell density and orientation in aged lymphatic wall¹⁶ are not able to preclude the ability of the aged MLV to be rapidly (within 5 minutes) stimulated, under resting conditions, up to the levels of pumping in the adult MLV. At the same time the question remains to be answered, “Will the rapid L-NAME (or any other drug)-induced stimulation maintain the levels of aged minute lymphatic pumping similar to the adult counterparts during the periods of the increased volumetric load (lymph formation) in aged body, and, if so, how long will it be maintained?” Moreover, the observations that L-NAME administration in vivo induced ∼2.5-fold greater increase of lymphatic pumping in comparison to isolated vessels moves us to the idea that in aged tissues surrounding the aged ML, the additional source of some yet unidentified metabolites which stimulate lymphatic contractions and whose effect may be counterbalanced or blocked by NO release. The post-L-NAME observed increases in diastolic wall shear stress (due to the vessels constriction, but not the diastolic lymph flow velocity) and minute active wall shear stress load (due to the increases in lymphatic contraction frequency) in aged MLV cannot be linked to the described differences between in vivo and isolated vessels since the NO synthase function was blocked during both experimental conditions. As a matter of fact, we believe that a focus for future follow up investigations on the nature of the aging-associated changes of pumping of the MLV to a large extent will relate to discovery of the mechanisms of potential interaction of the aged contractile lymphatic vessels and aged tissues surrounding them.

As a direct follow up of the studies with adult and aged MLV discussed above,³⁰ we recently investigated the potential presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV) which influence aged lymphatic function.³⁵ In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D2 synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and tumor necrosis factor-alpha) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin, followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P, anti-rat DNP Immunoglobulin E, peptidoglycan from Staphyloccus aureus, and compound 48/80) in presence of Ruthenium Red, followed by staining by toluidine blue. We found that there was 27% aging-associated increase in total number of mast cells, with ∼400% increase in number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that a higher degree of pre-activation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.

Aging is considered to be a chronic inflammatory process with a shift towards a proinflammatory cytokine profile in tissues that may account for numerous deleterious vascular changes associated with aging.^36–38 The increased state of pre-activation as well as increased number of mast cells in aged mesentery, which we confirmed in this study, indicates existence of chronic inflammatory environment in mesentery since activated mast cells would have released their preformed inflammatory mediators such as histamine, proteases, and cytokines such as TNF alpha. Such aging-associated chronic inflammatory environment, a sign of which is an increased state of activation of aged mast cells in close proximity to aged MLV, may be one of the important causes of alterations in lymphatic pump function in aged animals. Previously, we demonstrated that application of L-NAME in situ caused an abrupt and significant increase in contraction frequency of aged MLVs but not in isolated vessels.³⁰ This proves that in the immediate vicinity of aged MLV, there are some counterbalancing mediators which have effects opposite to the lymphatic inhibitory effects of NO in aged rats. Such counterbalancing effects do not exist in isolated MLV preparations²⁴ because carefully dissected lymphatic vessels do not have mast cells in their walls, and surrounding tissues are eliminated during vessel isolation procedure. Presence of increased numbers of activated mast cells by aged MLVs indirectly confirms the existence of the increased local concentrations of histamine and other mediators that are known to influence lymphatic contractility, and in particular are able to induce positive chronotropic effect on lymphatic vessels. These mediators have potential to further increase contraction frequency of aged MLV in time when the inhibitory effect of NO is removed by L-NAME administration.³⁰ In light of our present findings, there is in average ∼400% increase in number of activated mast cells in aged mesentery in addition to 27% increase in their density near aged lymphatic vessels, we may consider the cumulative action of mast cells mediators chronically released from activated mast cells in resting conditions as an important mechanism of aged-induced shifts in lymphatic contractile function. Further detailed investigations are necessary to determine how different mast cell-derived mediators and by which mechanisms influence function of the aged lymphatic vessels.

Although there has been a great deal of work done on inflammation in vascular aging, there is lack of sufficient research that may apply to the aging of the lymphatic system. In spite of a chronic inflammatory state, aging has been thought to involve immunosenescence with deficient innate as well as adaptive immune response.^39–42 Mast cells are considered to be the first line of defense against allergens and pathogens and thus are important initiators of innate immune response.^43–47 Various inflammatory and chemotactic molecules produced and released from mast cells in response to foreign allergens or pathogens help recruitment of other antigen presenting cells at the site of inflammation and participate in shaping the host adaptive response.^48–51 Historically studies on mast cells in aging have been limited to dermal and lung mast cells in context of studies on allergy and asthma.^52–54 In this study,³⁵ we have for the first time analyzed the aging-associated changes in mast cell number and their behavior in rat mesentery near lymphatic vessels. In this study, we also have compared the responses of mesenteric mast cells to different acute stimuli in adult and aged animals. We used known mast cell activators: substance P—an inflammatory neurotransmitter; anti-rat DNP IgE—an allergic mediator; peptidoglycan from Staphyloccus aureus (PGN)—a classical toll-like receptors 2 ligand, and compound 48/80—standard chemical activator to stimulate mast cells ex vivo in live mesenteric tissue containing MLV. Unlike in vitro experiments where the total number of cells is arbitrary, our current ex vivo preparation helped us to evaluate the number of pre-activated cells as well as number of intact cells available in the particular tissue bed to be newly activated by acute inflammatory stimuli. We found that, in spite of the fact that 24-mo-old rats had ∼27% increase in total mast cell number in the mesenteric bed, the number of intact cells available to react to the presence of acute stimuli is significantly decreased in aged rats. We predict that the presence of extensive degranulation of mast cells in basal conditions in 24-mo-old rats promotes a chronic inflammatory environment but the reduced availability of intact mast cells to react to acute noxious stimuli is one of the major contributory reasons of delayed immune response to acute inflammation in elderly. We discovered that a smaller fraction of previously nonactivated mast cells were activated as a result of either biological or chemical stimulation in 24-mo-old rats as compared to 9-mo-old animals. However, in the case of IgE stimulation, the diminished ability of aged mast cells to be activated (lower number of activated mast cells after treatment) also has its own impact on age-associated alterations in functional status of mast cells and in their acute response to acute stimulation. The mean pixel intensity as a result of treatment (change of intensity after treatment from before treatment conditions) is always significantly less in 24-mo-old rats compared to 9-mo-old rats. The presence of extensive degranulation in basal conditions in aged mesentery increased the mean pixel intensity of mesenteric mast cells after treatment in 24-mo-old rats as compared to 9-mo-old rats but the increase in mean pixel intensity as a result of activation due to treatment was always significantly more in young rats as compared to old rats. Taken together, our findings indicate that in aging, mesenteric mast cells have reduced ability to be activated by acute inflammatory stimuli. While deficient immune cell function has been reported in aging,^39–42 there is no detailed information on the aging-associated alterations of mast cells and how they affect the function of mesenteric lymphatic vessels. The limited number of aged mast cells located in the mesentery to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response. Further investigations will be able to answer the important questions on the mechanisms of such effects discovered in this study.³⁵

The free radical theory of aging suggests that there is a progressive reduction in NO production and a simultaneous increase in free radical production leading to endothelial dysfunction. Beckman and Ames suggested that oxidative stress is an important factor contributing to vascular dysfunction with aging.⁵⁵ Vascular aging is associated with both structural and functional changes that can take place at the level of the endothelium, smooth muscle cells, and the extracellular matrix of vessels. Aging is also correlated with increased oxidative stress and oxidative damage, and the endothelium appears to be an important source of superoxide anion (O₂^•-) in the vascular wall. Marin and Rodriguez-Martinez reported⁵⁶ that endothelial cells are vulnerable to oxidative stress due to their low antioxidant capacity. Aging-related endothelial dysfunction may involve mechanisms such as alterations in the antioxidant defense systems, increased oxidative injury, or both. Studies suggest that inactivation of nitric oxide by superoxide contributes to impaired vascular function.^57–60 NO reacts with superoxide radical (O₂^•-) to form peroxynitrite (ONOO^-), which can further induce protein modification and DNA damage in the microvascular system.⁶¹ Thus, decrease in NO bioavailability as a result of excess O₂^•- formation is a major cause of endothelial dysfunction in aging. Aging-associated elevations in oxidative stress may be related to alterations in antioxidant defense enzymes such as the superoxide dismutase (SOD) isoforms Cu/Zn-SOD (located in cytoplasm), Mn-SOD (located in mitochondria), and extracellular SOD (EC-SOD). Additionally, the oxidative stress may come from increased production of reactive oxygen species (ROS) via mitochondrial dysfunction, activation of NADPH oxidase, or uncoupling of nitric oxide synthase (NOS).^62–64 The balance between the levels of free radical production and cellular antioxidant activity determine the oxidative stress on the tissue and subsequent degree of oxidative damage. Importantly, Zawieja et al. reported⁶⁵ that oxygen radicals significantly inhibited contractile activity of rat mesenteric lymphatic vessels; thus we propose that aging-associated increases in oxidative stress could contribute to the decline in contractile activity seen in aged lymphatic vessels. Currently, there are no investigations of aging-associated oxidative stress or oxidative damage in lymphatic vessels. Thus, the goal of the one of our recently published work⁶⁶ was to examine the aging-induced changes in expression and activity of the major cellular antioxidant enzyme, superoxide dismutase (Cu/Zn-SOD, EC-SOD, and Mn-SOD isoforms) while also evaluating peroxynitrite-mediated cellular damage and mitochondria-related superoxide radical production in aged mesenteric lymphatic vessels from Fischer-344 rats in comparison with their adult counterparts.

We measured the total activity of SOD enzyme activity in adult and aged MLV and found a significant decrease in 24-mo-old MLV (3.98±0.08 Unit/mL) compared to 9-mo-old vessels (4.36±0.04 Unit/mL).⁶⁶ Additionally, we measured thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation and found that the concentration of TBARS was significantly elevated in 24-mo-old MLV (53.7±10.8 nM/mg wet tissue) when compared to 9-mo-old vessels (3.4±0.8 nM/mg wet tissue). Next we determined the total cellular superoxide and mitochondrial reactive oxygen species (ROS) production in mesenteric lymphatic vessels by measuring the total levels of cellular superoxide by DHE fluorescence in live adult and aged MLV. After 30 min of incubation with this superoxide dye, numerous fluorescently labeled cells were identified in MLV, and the density of fluorescent signal was significantly higher (∼97%) in the 24-mo-old MLV (24.7±2.9 arbitrary units) when compared to 9-mo-old vessels (12.5±0.8 arbitrary units). Additionally, we assessed mitochondrial ROS production using the mitochondria specific ROS-sensitive fluorescent dye MitoTracker Red CM-H2XRos and identified significantly elevated (∼61%) production of mitochondrial ROS in 24-mo-old MLV (21.3±1.9 arbitrary units) compared to 9-mo-old vessels (13.2±2.1 arbitrary units).

Furthermore, we performed Western blot analyzes to compare the protein expression of SOD isoforms (Cu/Zn- SOD, Mn-SOD, and EC-SOD) and nitrotyrosine (as an indicator of oxidative damage) in 9-mo-old and 24-mo-old MLV. We found that the Cu/Zn-SOD isoform was expressed significantly lower (∼51%) in 24-mo-old MLV (normalized signal intensity (NSI): 2.53±0.33) compared to 9-mo-old MLV (NSI: 5.16±0.53), while the EC-SOD isoform was expressed significantly higher (∼78%) in 24-mo-old MLV (NSI: 5.42±0.53) compared to 9-mo-old MLV (NSI: 3.04±0.40). We did not find significant differences in the expression of the Mn-SOD protein isoform between 9-mo and 24-mo-old MLV samples (NSI: 0.96±0.18 versus 1.60±0.31, respectively). Protein-bound nitrotyrosine formation is commonly used to demonstrate nitric oxide-dependent oxidative damage. In this study, we examined the quantity of nitrotyrosine formation in 9-mo and 24-mo-old MLV via Western blot. We observed a significant increase (∼148%) in the expression of nitrotyrosine protein corresponding to 215 kDa band in 24-mo-old MLV (NSI: 1.05±0.18) when compared to 9-mo-old MLV (NSI: 0.42±0.14). Conversely, we did not observe significant differences in the 36 kDa band between age groups.

We also performed immunohistochemical labeling of SOD isoforms (Cu/Zn- SOD, Mn-SOD, and EC-SOD) and nitrotyrosine (as an indicator of oxidative damage) in 9-mo and 24-mo MLV. We found that the signal intensity (relative to background) for Cu/Zn-SOD isoform was significantly lower (∼28%) in 24-mo-old MLV (relative signal intensity (RSI): 6.31±1.32) compared to 9-mo-old MLV (RSI: 8.73±1.64). At the same time, we did not find significant differences in the signal intensities of Mn-SOD and EC-SOD isoforms between 9-mo and 24-mo-old MLV samples (RSI: 9.93±4.86 versus 6.87±1.40 and 11.54±1.33 versus 11.87±2.67, respectively). We also examined the signal intensity of nitrotyrosine in 9-mo and 24-mo MLV via immunohistochemical labeling to demonstrate NO-dependent oxidative protein damage. We observed a significant increase (∼47%) in signal of nitrotyrosine labeling in 24-mo-old MLV (RSI: 10.67±0.44) when compared to 9-mo-old vessels (RSI: 7.24±0.43).

Aging is commonly defined as a functional loss over time that is accompanied by an inability to withstand stress or insult. We recently demonstrated that, as with many other organs, there is an aging-associated decline in the contractile capacity of lymphatic vessels.^4,24 However, the cellular events responsible for this functional loss remain undefined. The role of ROS such as O₂^•- in the aging process was initially proposed as the “free radical theory” by Harman in the 1950s,⁶⁷ whereby ROS damage the cellular constituents resulting in a functional decline of the organ systems finally leading to cell death. The participation of ROS is well documented in many pathological conditions typical for the elderly such as cardiovascular diseases, diabetes, cancer, and arthritis. In this present study,⁶⁶ we provided the first evidence for aging-associated elevations in mesenteric lymphatic vessel O₂^•- and oxidative cellular damage.

Endothelial cell membrane damage is thought to be an early event leading to microvascular dysfunction and may be initiated by several factors including lipid peroxidation. ROS, such as hydrogen peroxide, superoxide, and hydroxyl radicals, damage biomembranes and induce peroxidation of lipids, leading to an increase in cell permeability and loss of endothelial integrity. In this current study,⁶⁶ we observed a significant increase in the concentration of TBARS in mesenteric lymphatic vessels of 24-mo compared to 9-mo-old rats. We considered this observed increase in the levels of TBARS to be an indicator of elevated aging-associated free radical-induced lipoperoxidation in MLV that could ultimately be associated with cellular membrane damage. These findings correlate with observations by Ohkuma⁶⁸ who proposed that interstitial accumulation of lipoperoxide (a breakdown product of cell membranes) may be a potent toxic factor responsible for trophic changes associated with chronic lymphedema (a pathological condition also associated with reduced lymphatic contractility^69–71). In 1993, Ohkuma suggested⁷² that during the impaired lymph drainage, lipoperoxides, which are normally transported in lymph, may be deposited in the skin and contribute to the soft tissue changes characteristic of chronic lymphedema. In respect to Dr. Ohkuma findings and given the role of mesenteric lymphatics in transporting dietary lipids, we propose that aging-associated lipoperoxidative damage in the mesenteric lymphatic network observed in the present study and the impairment of the mesenteric lymph transport function in aged vessels reported by us earlier^24,30 may predispose the elderly to excessive mesenteric fat deposition, thereby potentially contributing to the lipid dysregulation commonly seen in aging-associated diseases. Potential inflammatory-related breakdown of antioxidant systems in mesenteric lymphatic vessels might be considered as an important trigger in the development of metabolic syndrome, even at earlier stages of the life span, which requires further detailed investigation.

As discussed above, diminished NO bioavailability due to increased O₂^•- production is one of the major mechanisms responsible for the impaired endothelium dependent vasodilator responses observed during aging. The interaction between NO and superoxide depletes NO bioactivity, thereby altering several key vascular functions of which NO is a pivotal mediator including regulation of smooth muscle tone, platelet activation, and vascular cell signaling.^73–75 In currently discussed study,⁶⁶ we report the first evidence of a significant increase in superoxide production in live aged mesenteric lymphatic vessels, thus confirming the existence of the aging-associated oxidative stress in MLV, which may be a key factor in the cascade of events occurring in the mesenteric lymphatic network as a biological consequence of aging.

In normal physiological conditions, antioxidant enzymes prevent the detrimental effects of O₂^•-. In studying the role of antioxidant defenses in aging, attention has been given to the role of superoxide dismutase, which efficiently and specifically catalyzes the dismutation of O₂^•- to H₂O₂ and O₂. SODs are also involved in the modulation of NO bioactivity. Normally, tissues express three isoforms of SODs including Cu/Zn-SOD (SOD1), Mn-SOD (SOD2), and extracellular SOD (EC-SOD or SOD3). Cu/Zn-SOD is an abundant copper- and zinc-containing cellular protein that is present in the cytosol, nucleus, peroxisomes, and mitochondrial inner membrane. Its primary function is to lower the intracellular steady-state concentration of O₂^•-.⁷⁶ Mn-SOD is a mitochondrial enzyme that disposes of O₂^•- generated by respiratory chain activity. It can be induced to protect against pro-oxidant insults. Conversely, Mn-SOD activity is decreased in physiologic aging and in diseases such as cancer, asthma, and transplant rejection.⁷⁷ EC-SOD plays an important role in regulating blood pressure and vascular contraction, at least in part through modulating the endothelial function by controlling the levels of extracellular O₂^•- and nitric oxide bioactivity in the vasculature.^78,79 Aging has been shown to produce alterations in the expression and activity of SOD in several tissues.^80,81 In blood vessels, SOD activity and/or expression has been shown to be altered with aging.^82,83 Zawieja et al.⁶⁵ reported that in young mesenteric lymphatic vessels, decreases in ejection fraction, contraction frequency, and lymph pump flow caused by high dose superoxide anion treatment were attenuated by SOD application. In our study,⁶⁶ we observed that total SOD activity was significantly decreased in the aged mesenteric lymphatic vessels compared to adult vessels. This result may be a contributing factor to the elevated ROS in aged vessels and/or may augment the oxidative stress on the vessels during aging, leading to elevated levels of oxidative damage. In parallel with data obtained by Zawieja et al.,⁶⁵ we propose that diminished SOD enzyme activity may play an important role in development of the aging-associated impairment of the mesenteric lymph transport function reported by us earlier.^24,30 However, further functional investigations are necessary to confirm the role of SOD protein dysfunction during aging in mesenteric lymphatic vessels.

The relative expression of different SOD isoforms in cells and tissues has been investigated extensively and provides clues as to the sources of O₂^•- in pathophysiologic states. Cu/Zn-SOD is the predominant isoform in microvessels where it may scavenge O₂^•- to increase the bioavailability of NO, which in turn improves endothelium-dependent vascular function. Didion et al.⁶⁰ suggested that the release of NO from the endothelium is dependent on Cu/Zn-SOD, whereas EC-SOD activity is thought to be required for the protection of NO as it diffuses through the vascular wall. In both large arteries and microvessels, deficiency in Cu/Zn-SOD results in increased levels of vascular superoxide and peroxynitrite, increased myogenic tone, augmented vasoconstrictor responses, and impaired endothelium-dependent NO-mediated relaxation.^59,84,85 In the current study, we confirmed by Western blot analyses and immunohistochemical labeling significantly lower levels of Cu/Zn-SOD isoform expression in 24-mo-old compared to 9-mo-old MLV; such changes may be a contributor to the aging-associated impaired endothelium-dependent NO-mediated regulation on the lymphatic vessels.^4,24,30 While we did observe an increase EC-SOD isoform expression in 24-mo-old compared to 9-mo-old MLV via Western blot, we were not able to see any difference in expression of EC-SOD via immunohistochemical labeling. While these differing results for EC-SOD may simply reflect the detection abilities of these two techniques, it is important to note that total SOD activity in 24-mo-old MLV is still decreased relative to the 9-mo-old vessels. Thus, even if EC-SOD protein expression is indeed elevated in the 24-mo-old vessels (as indicated via Western blot), the total antioxidant activity of the aged vessels is still depressed relative to the adult vessels, presumably due to the consistent depression in Cu/Zn-SOD protein expression. Follow-up studies are needed to assess the specific importance of the Cu/Zn-SOD isoform depletion and the possible EC-SOD enrichment in the development of the aging-associated oxidative damage in lymphatic vessels.

The increased levels of O₂^•- in aged MLV may result in increased NO scavenging and subsequent ONOO^- formation, which is known to initiate oxidative modification of proteins, ultimately leading to lipid peroxidation or DNA damage.^55,86,87 It is known that peroxynitrite at submicromolar concentrations causes the nitration of protein-bound tyrosine residues in Mn-SOD or prostacyclin synthase.⁸⁸ Our data clearly demonstrate significantly increased nitrotyrosine levels in aged mesenteric lymphatic vessels, thus indicating increased formation of ONOO^-. This last observation suggests that there may be diminished levels of basal NO in the vessel due to the inability of SOD to completely scavenge the elevated O₂^•- formation during aging. Consequently, the loss of NO bioactivity associated with increased vascular O₂^•- in the mesenteric lymphatic vessels during aging may play a potentially important role in the pathogenesis of lymphatic endothelial dysfunction. We also found that mitochondrial ROS increased in the aged MLV compared to their adult counterparts. This also might enhance the reaction between O₂^•- and NO. It is demonstrated⁸⁹ that protein tyr-nitration mediated by ONOO^-, increases with age and ultimately leads to the inhibition of mitochondrial energy production by causing site-specific lesions in the electron transport chain, in enzymes involved in the citric acid cycle, or in enzymes necessary for energy transfer. In connection with these findings, aging-associated weakening of lymphatic pumping and disturbances in its NO-dependent regulatory mechanisms^4,24,30 may be linked to increased nitrotyrosine levels in aged lymphatic vessels; such conclusion still requires in additional experimental confirmation.

In this issue, we are presenting the results of the detailed quantitative evaluation of potential aging-associated changes in muscle cell density in MLV that have never been performed previously (see Bridenbaugh et al., this issue). In this study, we performed detailed evaluation of muscle cell density in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo-old, 9-mo-old, and 24-mo-old Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream and above lymphatic valves possess the lowest density of lymphatic muscle cells. Most of the high muscle cell density zones exist downstream to the lymphatic valve. The muscle cell density of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cell density. We conclude that the low muscle cell density zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells that are positioned above and near lymphatic valves and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels.

In conclusion, by reviewing the recent research findings related to the nature and the mechanisms of aging-associated alterations of lymphatic contractility and flow, we believe that during the recent years we were able to expand the current knowledge in this field of lymphatic biology. Although first critical steps on the way of discovery of the aging-associated alterations of lymphatic contractility and pumping were already performed, these new findings open important horizons for new scientific endeavors. We hope that future continuation of the research efforts in this area will provide not only novel fundamental knowledge on the biology of lymphatic aging, but also will create a solid foundation for subsequent developments of the lymphatic-oriented therapeutic interventions during many diseases in the elderly.

Author Disclosure Statement

This work was supported in part by the National Institutes of Health (NIH RO1 AG-030578 and HL-094269) and by Texas A&M Health Science Center College of Medicine and Department of Medical Physiology. Dr. Gashev and Mr. Chatterjee have no conflicts of interest or financial ties to disclose.