The primary finding of this study is that angiogenesis in adult SHR mesenteric microvasculature is not impaired during the network remodeling post stimulation. Microvascular growth induced by exteriorization of the mesentery was characterized by growth in vascular area and increased capillary sprouts and vascular length density. As an indicator of angiogenesis accompanied by arteriogenesis, we observed increases in total arterial and venous (A/V) length and segments per vascular area. By 25 days post stimulation, vascularized area, vascular density, vessel segments and capillary sprouting in SHR networks were comparable to Wistar networks. Since the initial network size of SHR networks was smaller than the size of Wistar networks, the hypertensive networks experienced an increased growth rate during the early stage of the remodeling process. Evidence of an increased angiogenesis was also supported by increased vascular density in the SHR compared to Wistar tissues at 5 and 10 days and increased capillary sprouting at 10 days.
Comparison of the percent change in vascular density between time points also indicated altered rates of remodeling in the SHR. At 3 and 5 days post our tissue exteriorization stimulation, the percent change in vascular length density was increased in the SHR relative to Wistar networks supporting an increased initial rate of angiogenesis. By 25 days, SHR networks experienced an increased rate of vessel pruning or dropout. Our characterization of network growth over a time course of remodeling indicates that mechanisms associated with vessel loss contribute to rarefaction in hypertension.
Since microvascular rarefaction during hypertension has been attributed to impaired angiogenesis (8
), our observations over a time course of remodeling offer an alternative perspective and an explanation for inconsistent results in the literature. In 4-week-old SHRs, angiogenesis has been documented to be impaired in response to hind limb ischemia (45
) and increased in a subcutaneous fibrin gel chamber implantation model (19
). Kiefer et al. examined the angiogenic capacity of serum from SHR and Wistar-Kyoto (WKY) rats at 6 weeks and 12 weeks old (21
). The results showed that at the age of 5 months, the angiogenic potential of serum of SHR is decreased but not in 6-week adult stage. They proposed that in SHR, there is transient period in which the vascular growth stimulating capacity is smaller than control rats. While the inconsistent results could be attributed to different models, tissues, ages, our results suggest that differences in angiogenic metrics also depend on the end time point in the study. For example, in our study comparisons of vascular areas at 3, 5, and 10 days would suggest that angiogenesis was impaired in the SHR. However, this result is not consistent when comparing vascular area at 25 days. In contrast, comparing microvascular length density or the number of vessel segments at early time points might suggest that angiogenesis is increased in the SHR; yet again at 25 days this result would not be supported. Based on our evidence of increased pruning of vessels in later stage of network remodeling, decreased vascular density measurement comparisons in other hypertension angiogenic studies might be explained by measurements made at time points after any peak in angiogenesis.
The importance of examining angiogenesis in hypertension versus normotensive networks over the time course of growth is emphasized by our characterization of angiogenesis in response to another inflammatory stimulus via 48/80 induced mast cell degranulation. At the day 2 time point for 48/80 induced angiogenesis in this study, capillary sprouts per vascular area of SHR was increased compared to normotensive control and comparable at 10 days. In contrast, the vascular density between groups was comparable at day 2 and decreased in the SHR at day 10. The data from our 48/80 studies further support the notion that comparisons from the literature depend on the angiogenic metric and the specific time point. Additionally, the 48/80 stimulation results at day 2 and day 10 are inconsistent with the comparisons made at similar time points in our exteriorization model. While further investigations would be required to fully characterize the difference of angiogenic responses in the two models, these inconsistencies suggest that angiogenic response comparisons between hypertensive and normotensive groups might also be model specific.
In the SHR, systolic blood pressure begins to rise at 5–6 weeks of age and may reach 180 mmHg by 15 weeks old, while the pressure of a normotensive rat is around 130mmHg (23
). By this age, microvascular rarefaction has been documented in numerous tissues including skeletal muscle, cardiac muscle, kidney and the brain of SHR (22
). Microvascular rarefaction has also been documented in the SHR mesentery (4
). While mesenteric blood flow rates average about 10% of the cardiac output or 40ml/min/100 g in the human body (12
), a limitation associated with mesenteric tissue is its unknown function and contribution to overall resistance (9
). In 15–16 weeks old SHRs, microvascular rarefaction has been correlated with microvascular dysfunction in the mesentery including increased wall-to-lumen ratio, enhanced microvessel specific oxidative stress, elevated MMP levels, elevated microvascular tone, deficient leukocyte-endothelial interaction and extensive non-uniform endothelial cell apoptosis (20
). Because these characteristics and vessel growth mechanisms in mesentery are similar to those in other tissues, the rat mesentery represents a model system for examining the microvascular network growth during hypertension. Future studies will be required to determine if the angiogenic response is different in older or younger hypertensive animals.
The SHR represents a genetic model of essential hypertension and the different angiogenic responses in this study could be explained by related strain differences between SHR and Wistar animals. However, the importance of the local environment on vessel growth is supported by the ability of microvascular networks in the SHR to reacquire normal vessel densities compared to normotensive strains. Rarefaction has been reversed with antioxidant treatment (22
), MMP inhibition (48
), VEGF gene transfer (8
) or chronic hypoxia stimulation (47
). In addition, treatment with angiotensin converting enzyme (ACE) inhibitor and angiotensin 2 type 1 receptor antagonist can reverse functional capillary rarefaction in muscle and skin of SHR (45
). Collectively, these studies along with our results support that microvascular networks in the SHR are able to undergo microvascular remodeling in response to local changes in the environment.
The importance of the rarefaction phenomenon in humans with hypertension is supported by the documentation of a reduced number of vessels in various from hypertensive patients including skin (2
), conjunctival circulation (43
), intestine (41
) and skeletal muscles (17
). The ability of the microcirculation in hypertensive patients to undergo normal remodeling is supported by the reversal of rarefaction with antihypertensive therapies (3
). Still, the cause-effect relationship between rarefaction and elevated blood pressure remains debated and the recent characterization of rarefaction in the skin of borderline essential patients and normotensive offspring of individuals with essential hypertension suggest that microvascular rarefaction could be a primary indicator and contributor to elevated blood pressure (1
). These results suggest that pre-hypertensive therapies targeted at reversing rarefaction might represent alternative treatment strategies and emphasize the importance of investigations aimed at understanding the mechanisms of rarefaction. Our results suggest that an emphasis on the pruning process is necessary for the design of long lasting rarefaction reversing treatments. For adults with developed hypertension, the development of multi-combination based therapies targeted at blood pressure regulation and preventing local end-organ damage require understanding how microvascular rarefaction is linked to microvascular dysfunction. While this is not the objective of our study, this issue does motivate the need for more fully understanding rarefaction dynamics and the ability of hypertensive networks to respond to tissue remodeling stimulus.
This particular model of angiogenesis was selected because it produced a robust angiogenic response over a relatively short time course via inherent mechanisms in a tissue and without the invasion of a foreign material. Another advantage of this mesentery exteriorization model is that it reflects physiological tissue response inside the body (10
). While the exteriorization of the mesentery has been linked to mast cell activation and an increase of local histamine level (10
), the exact mechanisms involved in this model have not yet been identified. Still, we selected this model because it represented a multi-factorial stimulus that could be used to assess the angiogenic potential of microvascular networks. Our exteriorization model also allows us to examine a local angiogenic response. Because tissue windows outside the exteriorized mesentery region do not exhibit angiogenic responses (data not shown), we speculate that systemic blood pressure is not influenced. Future studies would be required to assess the impact of angiogenesis on local blood pressure per network.
In summary, our work suggests that impaired angiogenesis may not be a contributor to rarefaction during hypertension. Over the time course of microvascular network remodeling, we observed both apparent increases in initial growth and subsequent increases in vessel loss. These altered rates suggest that inconsistent results in the literature regarding whether angiogenesis is impaired during hypertension might be explained by comparisons between hypertensive and normotensive groups at different relative time points during the angiogenic response.