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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2003 as doi:10.1096/fj.02-0626fje. |
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Department of Medical Physiology, Cardiovascular Research Institute, College of Medicine-Texas A&M University System Health Science Center, College Station, Texas, USA
2Correspondence: Department of Medical Physiology, Cardiovascular Research Institute, College of Medicine-Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA. E-mail: dcz{at}tamu.edu
SPECIFIC AIMS
In most tissues, generation and regulation of lymph flow is uniquely dependent on fast/phasic and slow/tonic contractions of lymphatic muscle. To begin to understand the molecular basis of lymphatic muscle contraction, we correlated lymphatic contractile function with profiles of contractile proteins and their messages in rat mesenteric and thoracic lymphatics and compared them to the profiles seen in arterioles.
PRINCIPAL FINDINGS
1. Functional analyses of rat thoracic duct contractile activity
Local prevailing pressure gradients dictate the need for the lymph pump to produce lymph flow. In mesenteric lymphatics, regular intrinsic oscillatory contractions form the basis of a strong lymph pump and are critical to the generation of lymph flow. However, the thoracic duct is exposed to a very different physical environment. Thoracic duct exhibited oscillatory phasic contractions that appeared to be involved in the pumping of lymph. The phasic contractions seen in the rat thoracic duct were less regular than those seen in the mesenteric vessels and did not propagate as often. The average contraction frequency of the thoracic duct (4.7 cpm) was 
one-half that in mesenteric lymphatics. The strength of the thoracic duct contraction was 60–75% of that in the mesenteric lymphatics. The strength of the contractions fell rapidly as transmural pressure raised above 1 cm H2O, in contrast to what was seen in other strong lymph pumps. The fractional pump flow of the thoracic duct was four- to fivefold lower than the mesenteric lymphatics. In general, these data indicate that the rat thoracic duct exhibits phasic contractile activity; however, the pumping effectiveness is significantly lower than that seen in rat mesenteric lymphatics.
2. Expression of myosin heavy chain isoforms in lymphatic muscle
The function of different muscle types is well correlated with the relative content of various MHC and actin isoforms. Mesenteric lymphatics expressed mRNA for only the SMB isoform of smooth muscle myosin heavy chain (SM-MHC) whereas the thoracic duct and arterioles expressed the SMA and SMB transcripts. Cultured lymphatic muscle cells (LMC) expressed only the SMA message. SM1 and SM2 isoforms of SM-MHC transcripts were both detected in all tested tissues. In LMC only SM1 messages were detected. Slow skeletal/fetal cardiac muscle-specific ß-MHC mRNA was detected only in mesenteric lymphatics. MHC RT/PCR data are summarized in Fig. 1
. The MHC protein profiles were studied using SM2, SMB, and slow skeletal/fetal cardiac ß-MHC-specific antibodies in Western blots and immunohistochemistry. Western analyses for lymphatic and arteriole samples demonstrated SMB and SM2 protein isoforms; in LMC there were no detectable reaction with either of these antibodies. Expression of slow striated MHC protein was only seen in mesenteric lymphatics. Protein expression using immunohistochemistry correlated with the Western blot results.
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3. Expression of actin isoforms in lymphatic muscle
All four actin messages (
-cardiac, -vascular, 
-enteric, and -skeletal) were present in mesenteric lymphatics and arterioles. However, the proportion of cardiac actin content appeared higher in mesenteric lymphatics than in arterioles. In the thoracic duct, -vascular and -cardiac actin messages predominated, with low-level expression of -enteric actin. In contrast, vascular, enteric, and skeletal actin transcripts were detected at higher levels in LMC; cardiac actin was found at very low levels. Using Western blot analysis, smooth muscle -actin, smooth muscle -enteric actin and cardiac/skeletal actin isoforms were found in all three lymphatic samples. However, the band detected in the thoracic duct represents cardiac -actin, given the absence of skeletal -actin transcripts in this sample. Conversely, in LMC the band must represent skeletal -actin. In arterioles, vascular and enteric actins were present, but sarcomeric actins were not detected. Immunofluorescent staining using these antibodies corroborated these findings.
4. Electron microscopy analysis of lymphatic vessel structure
Previous functional studies and the molecular analyses presented here demonstrate that the profile of contractile elements in lymphatic muscle cells is different from most vascular smooth muscle cells (VSMC). To evaluate the general architecture of the contractile elements in the lymphatic muscle cells, we performed transmission electron microscopy on isolated lymphatics. The muscle cells of the rat mesenteric lymphatic vessels appeared to be largely filled (70–80%) with an organized array of filaments. This arrangement is different from the organization of contractile filaments observed in typical VSMC. Given their size, shape, and prevalence, this highly structured array of the filaments seen in the EM matched the appearance of the contractile filament staining observed in our fluorescence staining studies.
CONCLUSIONS AND SIGNIFICANCE
The lymphatic system plays important roles in fluid and macromolecular homeostasis, immune function, and lipid absorption, relying on a regulated flow of lymph to do so. Lymphatic vessels must both generate and regulate the lymph flow. Thus lymphatics act not only as conduits of lymph, but also serve as the lymph pump. The lymphatic system relies on lymphatic muscle contraction to fulfill this unusual combination of roles. The cellular basis of lymphatic muscle contraction and how it compares with that found in other muscle cell types are poorly understood. These results are the first description of the contractile proteins found in lymphatics. The profile of contractile proteins in the lymphatics is quite different from that found in arterioles, presumably related to the large functional differences in these vessels. Lymphatic contractile activity consists of tonic and phasic contractions. Tonic contractions regulate lymph flow by altering lymphatic outflow resistance similar to the tonic contractions in blood vessels that regulate blood pressure and flow. Rapid phasic lymphatic contractions generate the pressure needed to open and close the appropriate valves and produce lymph flow. These phasic contractions are a unique vascular characteristic critical to lymphatic function. Generally, the rapidity of lymphatic contractions is much faster than that observed in arterioles (note the difference in the time axes in Fig. 2
). Thus, it is logical that lymphatics have contractile proteins associated with faster muscle types. The data also show that different regional lymphatics exhibit different functional characteristics that are correlated with differences in their contractile proteins.
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The rat thoracic duct exhibited oscillatory phasic contractions that appear to be involved in the pumping of lymph. The spontaneous contractions seen in the rat thoracic duct are generally much less regular than those seen in the mesenteric vessels and do not propagate as often. The pumping effectiveness of the thoracic duct is four to fivefold less than the mesenteric lymphatics. The thoracic duct is a weaker, less efficient lymph pump. However, the thoracic duct is exposed to a very different physical environment than the mesenteric vessels are and presumably has a different role in the overall transport of lymph. The thoracic duct is the final outflow path of lymph to the venous blood and is exposed to the oscillations in pleural pressure associated with breathing, blood pressure pulsations within the aorta, and contractions of the esophagus. The results of this and other studies indicate that the thoracic duct is principally a conduit vessel that exhibits pumping activity at low lymphatic pressures and flows. This is distinct from the mesenteric lymphatics, which are much nearer to the input side of the lymphatic tree and are known to exhibit strong pumping activity that is needed to generate lymph flow.
The diversity of contractile protein isoforms, especially MHC and actin, has been well correlated with the function of muscle types. Studies of the molecular nature of muscle demonstrate the presence of four SM-MHC isoforms. The SM1 and SM2 isoforms differ in their carboxyl ends, whereas alternative splicing at the region of the ATP binding pocket generates the SMA and SMB variants. Several studies have demonstrated that the SMB isoform is expressed in smooth muscle types with faster contractile properties, such as the bladder. It has a nearly twofold higher ATPase activity and it is proposed that the rate of force generation with the SMB isoform is greater than that by SMA. Because of the phasic contractile nature of lymphatics, we anticipated the expression of SMB MHC isoform in these tissues. Our data demonstrated the presence of SMB MHC in MLM and TLM. Our results indicated that another faster nonsmooth muscle MHC isoform, slow skeletal/fetal cardiac ß-MHC, was also present in MLM tissue. Thus, we found that the lymphatic tissues that exhibited stronger pumping activity also predominantly expressed the faster MHC isoforms.
In both lymphatic tissues, sarcomeric actin messages and proteins are present along with the vascular smooth muscle actin. It has been shown that the highly conserved individual muscle actins are functionally specialized for the tissues in which they predominate. However, flexibility in the genetic reprogramming of the actin gene family has been demonstrated in knockout mouse models. It is intriguing that the differential actin gene expression is seen naturally in the lymphatic system, yielding an interesting model of contractile protein gene expression.
Taken together, the molecular data provide strong evidence for the nature of the lymphatic vessels’ functional similarities to vascular smooth and cardiac muscle cells. Our EM data show a somewhat heterogeneous but highly organized appearance of contractile filaments in the MLM tissue. The array of filaments appears to fill 70–80% of the muscle cell section. These filaments were arrayed with a lateral displacement of 60 nm, similar to what has been described in rabbit portal vein, another vessel where there is organized, oscillatory phasic contractions related to the generation of flow within that vessel. The contractile elements are more organized in these mesenteric lymphatics than what is observed in typical vascular smooth muscle. These results strongly support our hypothesis that within different lymphatic beds, regional variations occur both in function and the contractile elements.
In conclusion, our data demonstrate that lymphatic muscle cells have unique contractile machinery that has both smooth and striated muscle components (Fig. 3)
that fit the unique roles of the lymphatic vessels. There are variations in the contractile proteins among two different lymphatic beds, the mesenteric lymphatics, and the thoracic duct. In addition, striking functional differences are seen within these two lymphatic tissues. Although it is premature to determine cause and effect, it is noteworthy that the molecular diversity in the contractile protein genes present within the lymphatic beds, as well as between the lymphatic and vascular tree, fulfills the characteristic functional differences that exist among these tissues (Fig. 3)
. This work establishes a basis for better understanding the lymphatic contractile process; future work may shed more light on the roles of different isoforms of contractile and regulatory proteins in lymphatic muscle function.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0626fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0626fje 
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X-174 DNA is used as marker. Left: positions of the molecular weight marker; right: expected positions of the PCR products