Invited Symposium: Hypertension III: Flow-Induced Vascular Remodeling



Materials & Methods


Discussion & Conclusion



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Nitric Oxide Synthase and Growth Factor Expression in Flow-Induced Arterial Remodeling in Rat Mesenteric Vasculature

Contact Person: David A. Tulis (DAMIT@RUF.RICE.EDU)

Discussion and Conclusion

Existing studies have demonstrated that arterial caliber and morphology can be modulated by blood flow and associated wall shear forces (1,2,7,8,10,11). However, many aspects of acute responses as well as chronic adaptations of arteries to blood flow and its associated stresses remain unclear. The current study utilizes a new experimental model to provide significant insight regarding how the arterial wall accommodates rapid luminal expansion and medial wall remodeling from exposure to elevated blood flow over a 7 day period. This model allows two arteries (ileal and second-order branch) to be exposed to varying degrees of flow elevation within the same animal without the influence of altered local or mean arterial pressures (Table 1). Results demonstrate that flow-mediated vessel wall structural remodeling is manifested as lumen expansion and medial wall hypertrophy, and the magnitude of change of these parameters is dependent on the duration of flow elevation after abrupt arterial occlusion. In addition, results suggest that EC and SMC hyperplasia concomitant with increases in ECM-associated constituents characterize this response. The influences of NO via eNOS upregulation, and the mitogenic contribution of PDGF-A, are inferred as possible mediators of the flow-induced signal transduction pathway.

An important observation in this study was that during rapid expansion of the arterial lumen, the wall to lumen relationship in the flow-loaded arteries did not deviate significantly from that observed in the control arteries at each time point. This suggests that parameters controlling lumen enlargement and medial wall hypertrophy are tightly regulated in these vessels. The exact mechanisms responsible for this are not clear. As supported by other studies (4,19,20), we consider the major initial stimulus for vessel remodeling in these arteries to be elevation of stresses associated with increased blood flow. Acute flow-induced dilation in the ileal artery (~7%) neutralized significant increases in calculated average WSR and may indicate a minor influence from stretch-induced hoop stress. The second-order artery, however, did not significantly vasodilate upon exposure to increased flow resulting in a highly significant doubling of its calculated WSR. We believe that the forces related to blood flow evidenced in this study in both ileal and branch vessels are primarily shearing forces with possible augmentation by stretch-induced hoop stress in the ileal artery.

In the experimental arteries, blood flow was initially increased 36% and 170% respectively in the ileal artery and second-order branch. Inner lumen diameters increased by ~13% at day 7 in the ileal artery while the second-order branch which experienced greater increases in flow was significantly enlarged ~14% by day 3 and ~40% by day 7. In addition, the second-order branch experienced greater percent changes in medial wall area when compared to the ileal artery over the 7 day period. In addition, both lumen expansion and medial wall hypertrophy experienced by the second-order branch were determined to be highly dependent on the duration of flow exposure between 1 and 7 days, whereas the ileal artery experienced a significant dependency on time only for increases in medial wall area. Although both order vessels exhibit structural remodeling resulting from elevated blood flow, the heightened responses in the second-order branch may indicate a dependency on the degree of flow elevation for flow-induced remodeling.

The significant direct influence of blood flow on intimal EC replication is also observed in this study. Lumen enlargement demonstrated in the high flow vessels was associated with significant EC proliferation. Intimal ECs were rapidly stimulated to replicate from exposure to elevated blood flow as shown by significant EC hyperplasia after 24 hours (Fig. 5). In addition, EC density increased significantly in a time-dependent fashion upon exposure to increased flow (Fig. 5B). This may be part of an adaptive process aimed at maintaining a constant intimal EC density coincident with luminal enlargement. These results are consistent with the recent findings of Unthank et al (10), who showed the number of EC nuclei in high flow mesenteric collateral vessels was ~90% greater than those found in control vessels after both 1 and 4 weeks.

Results from immunostaining for PCNA (Fig. 3), a cell cycle-dependent cyclin, suggest ileal and second-order branch medial SMCs undergo significant DNA replication after 3 and 7 days of in vivo exposure to elevated blood flow. Polyploid cells with DNA replication in the absence of cytokinesis, however, can erroneously be counted positive according to PCNA data. Absolute medial SMC nuclear counts (Fig. 4A) suggest cellular hyperplasia after 7 days of elevated flow in both the ileal and second-order arteries. These data imply that elevated flow stimulates DNA replication and cellular division in medial SMCs under physiologic conditions. Normalized SMC nuclear densities for both ileal and second-order arteries (Fig. 4B) supports a role for medial SMC hyperplasia contributing to medial wall hypertrophy in response to elevated flow. Considering the second-order vessel after 7 days, however, medial SMC hypertrophy and/or nuclear polyploidy may be occurring with numerous SMCs actively engaged in the cell cycle prior to cytokinesis as supported by the PCNA data. Once the remodeling process becomes complete, cellular hyperplasia and diploidy would subsequently result.

Another important observation from this study was that as the medial wall hypertrophied in response to flow elevation, synthesis of medial extracellular connective tissue increased resulting in a constant percentage connective tissue at all times. The increase in connective tissue may help maintain normal cell-cell contact concomitant with an enlarged wall area in an effort to maintain normal vessel geometry. Alterations in extracellular matrix constituents after exposure to flow has been previously demonstrated by several investigators (21,22).

A major finding of this study is the highly significant upregulation of medial wall PDGF-A mRNA expression in the ileal artery after exposure to increased blood flow for 24 hours. To our knowledge, this is the first report identifying growth factor transcript expression in response to flow elevation under in vivo physiologic conditions. One hypothesis for flow-mediated arterial remodeling involves shear stress-induced vasodilation leading to elevated circumferential hoop stress in the medial wall. This hoop stress, then, can stimulate transcription and release of medial smooth muscle-specific growth factors. Immunocytochemical data from our laboratory (not included) demonstrate significant upregulation of eNOS in arteries exposed to increased flow. Nitric oxide-mediated vasodilation and subsequent elevation of circumferential hoop stress could, then, stimulate synthesis of medial wall PDGF-A mRNA, as evidenced in our study. Similar results were reported by Wilson et al. (23), who showed elevated PDGF-A mRNA expression in cultured SMCs exposed to cyclic stretch after 24 hours.

Autocrine mitogenicity from PDGF-A, as suggested by several investigators (24,25), and paracrine action, as proposed by Vlodavsky et al. (26), may both be inferred in the current model. As observed, early medial PDGF-A expression precedes subsequent medial SMC DNA synthesis, cellular hyperplasia, and resultant medial wall hypertrophy. In addition, elevated PDGF-A transcript expression in the medial wall may be contributing to neighboring EC replication, which is evident after exposure to elevated flow starting after 24 hours. Nevertheless, extrapolation from enhanced transcript expression to functional protein involvement requires strict prudence, and post-translational modification as well as growth factor receptor modulation must be considered. Results showing no significant differences in growth factor expression between control and high flow second-order branches, however, seem counter-intuitive. Heterogeneity in vascular growth responses between different orders of vessels as well as between components of a single vessel has been characterized by several investigators (27,28), and may be present in the rat mesenteric small arteries examined in this investigation.

If the medial wall PDGF-A mRNA expression observed in this study correlates with enhanced protein translation, then PDGF-A may be acting through an autocrine mechanism to stimulate synthesis of medial ECM components observed in this study. The ECM is capable of regulating storage and release of growth factor proteins (29). These peptides, including PDGF and bFGF, may then be sequestered in the surrounding ECM with subsequent release regulated by ECM-associated proteolytic enzymes (29).


In conclusion, this report further characterizes arterial remodeling subsequent to blood flow increases using a unique in vivo physiologic model that allows different degrees of flow elevation within the same animal. This model analyzes the early sequence of events and dependency of arterial remodeling on the duration of flow elevation over a 7 day period. The primary stimulus in this model is believed to be flow-mediated shear stress with possible contribution from stretch-induced hoop stress. Significant lumen expansion and medial wall hypertrophy occurred in a time-dependent fashion after flow elevation for 7 days. These adaptations involved significant SMC and EC hyperplasia and ECM restructuring. Early medial expression of PDGF-A mRNA and stimulated eNOS expression in high flow arteries suggest a possible signaling mechanism involving flow-mediated NO-dependent vasodilatation resulting in increased circumferential hoop stress. This, in turn, could stimulate early medial wall expression of PDGF-A mRNA and positively influence cell cycle kinetics of both SMCs and ECs through autocrine and paracrine mechanisms. A reduction shear stress at the arterial wall after 3 days could contribute to delayed endothelial expression of PDGF-A mRNA. Temporal regulation of PDGF-A is also suggested, as significant medial expression after 24 hours precedes endothelial expression after 3 and 7 days.

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Tulis, DA; Unthank, JL; Prewitt, RL; (1998). Nitric Oxide Synthase and Growth Factor Expression in Flow-Induced Arterial Remodeling in Rat Mesenteric Vasculature. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/prewitt/tulis0444/index.html
© 1998 Author(s) Hold Copyright