We interrogated the physiology of cerebral vasculature in order to identify differences in functional activity between tumor-derived vasculature and the native vasculature from which it sprouted. Specifically, we quantified the reactivity of both total and microvascular CBV to alterations in PaCO2. Although vasodilation was blunted significantly in glioma vascular bed as compared to normal striatum, tumor vessels did constrict effectively in response to hypocarbia and to a greater extent than the response of normal subcortex.
These results demonstrate several important features regarding angiogenesis in experimentally derived tumors. Firstly, although tumor vascularity may differ significantly from the tissue of origin, blood vessels developed as a result of tumor angiogenesis can exhibit functional activity typical of the tissue from which the vessels sprouted. At normocarbia, blood volume in the tumors examined here was significantly elevated relative to surrounding brain and contralateral normal striatum. In addition, the elevation in the total blood volume relative to normal brain was significantly larger than the elevation in the microvascular compartment alone; total blood volume was elevated by 51% more than microvascular blood volume in gliomas. This indicates a disproportionate increase in blood contained within larger vessels in the tumor vascular bed. This derangement of the tumor vascular bed observed with imaging agreed well with the histological analysis that confirmed the significantly more vascular nature of these gliomas relative to normal striatum. Fractional vascular area and vessel number in gliomas were increased 2.4- and 2.2-fold, respectively, relative to normal striatum. These values are comparable to the 2.5-fold increase in microvascular blood volume determined by MRI. Moreover, the distribution of tumor vessel diameters revealed a pronounced shift in the population to larger vessels; mean vessel diameter was increased by 63% in tumors relative to striatum, confirming the disproportionate increase in blood volume contained within larger vessels in tumors. These results also agree well with previous reports of elevated blood volumes in experimentally implanted gliomas and the dilated and tortuous blood vessels [4
] that characterize the three-dimensional vascular network [2,25
]. Nonetheless, despite these distortions of normal vessel anatomy and vascular architecture, we demonstrated an appropriate functional response of the U87 glioma vascular bed to both dilate and constrict with an acute change in Pa
Secondly, although the response of the tumor vascular bed to hypercarbia was significantly blunted as compared to normal tissue, this did not translate into a reduction in functional ability to constrict in response to hypocarbia. Previous reports have described little or no increase in tumor BOLD signal intensity in experimentally implanted tumors challenged with hypercarbia [10–12
]. Neeman et al. [11
] proposed that the differential response in BOLD contrast between tumors subjected to hypercarbia alone and hypercarbic hyperoxia could be used as a noninvasive technique to identify immature vessels that lack the functional activity to dilate. Although we, as did they, observed impaired vasodilation, our demonstration of highly effective vasoconstriction within the tumor bed contradicts the notion that the blunted response to hypercarbia is due to vessels within the tumor that lack functional activity and, therefore, can be classified as “immature.” Although these results do not rule out the presence of a subpopulation of nonfunctional vessels present in the tumor, they do point out that the gross response of the vasculature to a single physiological challenge should be used with caution to comment on the microanatomy of the vascular bed.
Further evidence arguing against a preponderance of small nonfunctional blood vessels within the vascular bed is the good agreement in reactivity between the total blood volume and microvascular blood volume compartments. Because relative CBV determined by GE imaging is sensitive to all blood vessels, whereas SE imaging is primarily sensitive to vessels less than approximately 30 µm, it is possible to distinguish blood volume compartments with MRI [18
]. If, as has been proposed, the tumor vascular bed contained a large population of “immature” blood vessels that lack structural elements necessary for vasoreactivity, then the microvascular compartment that contains predominantly these smaller “immature” vessels would be expected to exhibit less reactivity than the total blood volume compartment compartment that contains larger, more reactive vessels. Similar to the behavior of normal brain, however, there was no significant difference in reactivity between blood volume compartments. Tumors derived from the C6 glioma cell line also have a comparably disproportionate increase in blood volume contained within large vessels [2
] as demonstrated for the U87 cell line reported here. Therefore, vessel size distribution cannot account entirely for the difference in results reported here and those of Abramovitch et al. [10
] and Neeman et al. [11
]. It is also important to note, however, that previous studies have indicated only a minor contribution of capillaries to adjustments in blood volume produced by moderate hypercarbia [27–29
As has been reported previously [2,20,30
] and confirmed here, we found no significant difference in reactivity to carbon dioxide between total and microvascular blood volume compartments in normal brain. Therefore, the mechanical activity responsible for alterations in vascular tone within tissues affected both blood compartments equally. Because the reactivity to carbon dioxide was also similar for both blood volume compartments in gliomas, we conclude that this physiological function was also preserved during angiogenesis. Despite the disproportionate increase in blood volume contained within large vessels and distortions in normal vessel anatomy, the mechanisms necessary for normal maintenance of vascular resistance appear intact in these tumors. Therefore, the tumor vascular bed appears to function more like normal brain than would be expected based on the gross differences in vasculature.
There are two previous reports of tumor vasculature that retain reactivity to changes in Pa
. Robinson et al. [13
] attributed a 10–30% increase in T2*-weighted MRI signal intensity during hypercarbia to be consistent with an increase in tumor blood flow resulting from vasodilation in GH3 prolactinomas. Cenic et al. [31
] reported significant decreases in both blood flow and blood volume in VX2 tumors implanted intracranially in isoflurane-anesthesized rabbits during hypocarbia. Interestingly, neither normal brain nor tumor demonstrated any significant decrease in CBF or CBV with hyperventilation when rabbits were anesthesized with propofol. Although the specific characteristics of the C6 glioma cell line and the angiogenesis-derived tumor vasculature may have been responsible for the lack of response to hypercarbia in tumor xenografts implanted subcutaneously in nude mouse flank reported by Abramovitch et al. [10
] and Neeman et al. [11
], the limitations of BOLD imaging to detect small changes in physiological parameters may also have played a role. Alternatively, anesthetic conditions, baseline blood volume, or location of implantation may have contributed to the lack of response.
Several possible explanations could account for reduced vasodilation but normal vasoconstriction in glioma vasculature observed here. In their native state, tumor capacitance vessels could be already near maximally dilated and have little capacity to increase the diameter further in response to an increase in CBF. Alternatively, local glioma CBF may be near maximal with little capacity to increase. CBF has been shown to be elevated in this tumor model [32
]. Because the majority of tissue blood volume is located in the capillary and venous compartments and is under relatively low pressure, physical constraints of the immediate tumor environment could influence venous compliance as well. The presence of a space-occupying lesion within the closed confines of the cranium can increase intracranial pressure—a condition that impacts on cerebral hemodynamics. Elevation of tumor intrinsic tissue pressure could exert a similar effect [33,34
]. Finally, vascular reactivity could be a characteristic of individual tumor cell lines. Using the same experimental design described here, we quantified the vascular reactivity to carbon dioxide for tumors derived from the human glioma cell line GLI36. In that case, the increase in total and microvascular blood volume induced by hypercarbia was not different from the contralateral normal striatum (unpublished observation). Simultaneous determination of changes in CBF and CBV during physiological challenge could be helpful to distinguish between these possibilities.
The reactivity of CBV to hypercarbia for normal striatum reported here (1.5 ± 0.2% CBV/mm Hg Pa
) compares favorably to results obtained previously by others. Payen et al. [35
] reported a 1.7 ± 0.3% increase in CBV per millimeters mercury Pa
in the striatum for transition to hypercarbia over a similar range of Pa
using GE planar MRI in halothane/nitrous oxide-anesthesized normal rats. Similarly, Mandeville et al. [20
] reported a 1.3% increase and Zaharchuk et al. [30
] a 1.8% increase in CBV per millimeters Hg Pa
in the whole brain of halothane-anesthesized rats by GE MRI. For transition to hypocarbia, results are more variable in quantifying the reduction in total CBV. Using dynamic contrast-enhanced CT to measure CBV, Cenic et al. [31,36
] reported no significant difference in CBV during transition to hypocarbia in propofol-anesthesized rabbits, but a significant decrease of 1.1 ± 0.5% CBV/mm Hg Pa
under isoflurane anesthesia. This difference in reactivity was attributed to the vasoactive properties of the anesthetic to effect baseline values of CBV. Similarly, Weeks et al. [37
] found that reactivity to carbon dioxide varied widely depending on anesthetic conditions; although halothane and pentobarbital resulted in no significant change in CBV with hypocarbia, CBV decreased by 1.4% mm Hg Pa
under isoflurane anesthesia. In good agreement with these results, we also found no significant decrease in CBV in normal brain during hypocarbia under halothane anesthesia.
Among the functional properties that have been described for normal cerebral circulation, MRI has been reported to assess reactivity to carbon dioxide, autoregulation of blood flow, blood flow metabolism coupling, and integrity of the blood-brain barrier. Although penetration of the blood-brain barrier by small-molecular-weight contrast agents is used routinely to image neoplasms, the noninvasive determination of disruption of other physiological properties of tumor vasculature has not become common in clinical practice. Recent evidence has suggested, though, that neuroaxis neoplasms may be graded for malignancy noninvasively based on metabolic rate [38,39
], blood volume, and blood flow [40–42
], and the kinetics of permeability to contrast agents [43,44
]. Further studies are necessary to determine the value of vascular reactivity as a useful clinical marker either of malignancy or of degree of neovascularity. The availability of superparamagnetic intravascular susceptibility contrast agents that do not penetrate a disrupted blood-brain barrier will greatly facilitate the assessment of tumor vascular function in the clinical setting. Although it is unlikely that imaging will replace surgical biopsies in the near future, imaging of the functional properties of tumors may aid in the selection of biopsy sites to improve the accuracy of diagnosis and selection of therapy. Finally, MRI assessment of vascular function may help in elucidating the mechanism of angiogenesis and prove useful in the development and evaluation of antiangiogenic pharmaceuticals.