Purpose: Given the complex tumor microenvironment, targeting multiple cellular components may be the most effective cancer treatment strategy. Therefore, we tested whether antiangiogenic and immune-based therapy might synergize by characterizing the activity of DC101, an antiangiogenic monoclonal antibody specific for vascular endothelial growth factor receptor-2 (VEGF-R2), alone and with HER-2/neu (neu)^targeted vaccination. Experimental Design: Neu-expressing breast tumors were measured in treated nontolerant FVB mice and immune-tolerant neu transgenic (neu-N) mice. Neu-specific and tumor cellŝ pecific immune responses were assessed by intracellular cytokine staining, ELISPOT, and CTL assays.Results: DC101 decreased angiogenesis and increased tumor cell apoptosis. Although DC101 increased serum levels of the immunosuppressive cytokine VEGF, no evidence of systemic immune inhibition was detected. Moreover, DC101 did not impede the influx of tumor-infiltrating lymphocytes. In FVB mice, DC101 inhibited tumor growth in part through a T cell^dependent mechanism, resulting in both increased tumor-specific CD8 + T cells and tumor regression. Combining DC101 with neu-specific vaccination accelerated tumor regression, augmenting the lytic activity of CD8 + cytotoxicTcells. In tolerant neu-N mice, DC101only delayed tumor growth without inducing frank tumor regression or antigen-specific T-cell activation. Notably, mitigating immune tolerance by inhibiting regulatoryTcell activity with cyclophosphamide revealed DC101-mediated augmentation of antitumor responses in vaccinated neu-N mice. Conclusions: This is the first report of DC101-induced antitumor immune responses. It establishes the induction of tumor-specificT-cell responses as one consequence of VEGF-R2 targeting with DC101. These data support the development of multitargeted cancer therapy combining immune-based and antiangiogenic agents for clinical translation.

The effect of transmural pressure on the responsiveness of vascular smooth muscle was studied using rats with chronic occlusion of one external iliac artery. The arterial pressure in the occluded leg was reduced to approximately half of that in the contralateral unoccluded leg. Helical strips from the low-and high-pressure femoral arteries of spontaneously hypertensive rats and rats with deoxycorticosterone acetate-induced (DOCA) hypertension were compared with corresponding tissues from normotensive controls. The sensitivity of both low-and high-pressure artery strips from the spontaneously hypertensive rat was greater than that of control? when strontium or lanthanum was used as the agonist. The sensitivity of strips from both low-and high-pressure arteries from the DOCA-hypertensive rat was greater than that of controls when potassium, epinephrine, or calcium was the agonist. There was no difference in sensitivity between strips from the low-and high-pressure arteries in any group of rats. Maximum contractile force (contractility) was reduced in femoral artery strips from both legs of all hypertensive rats. The KCl-induced contraction of vascular smooth muscle from both femoral arteries of either form of hypertensive rat was not as readily depressed by high calcium concentrations as was that from the normotensive rat. Changes in sensitivity and contractility associated with hypertension could not be reversed by lowering blood pressure in one leg of a spontaneously hypertensive rat or prevented by protecting one leg from high pressure prior to the induction of DOCA hypertension. The altered sensitivity and contractility of arterial strips in these models of hypertension are not, then, secondary to the increase in wall stress. KEY WORDSsmooth muscle contractility smooth muscle sensitivity spontaneously hypertensive rats potassium DOCA-hypertensive rats lanthanum strontium calcium depression of KC1 contraction epinephrine• Extensive evidence from studies of perfused vessels (1) or vascular beds (2-7) as well as from studies of isolated vascular smooth muscle (8)(9)(10)(11)(12)(13) indicates that vascular responsiveness is increased in hypertension. A reasonable inference from this evidence is that the increase in responsiveness causes the elevated total peripheral resistance that results in hypertension. However, in view of recent findings showing that the increased wall stress of hypertension can produce profound adaptive changes in the artery wall (5, 14-16), more definitive evidence is needed to determine whether the increased responsiveness of vascular smooth muscle in the hypertensive rat occurs in the absence of increased wall stress. This study was supported in part by U. S. Public Health Service Grant HL-03756 from the National Heart and Lung Institute. Dr. Hansen was supported by NIH Training Grant GM-00353.Dr. Hansen's present address is Department of Physiology, Harvard Medical School, Boston, Massachusetts 02155.Received March 14, 1974. Accepted for publication February 12, 1975. This stu...

The functional orientation of vascular smooth muscle in the walls of aortae of DOCA-hypertensive and normotensive rats was determined by measuring the maximum force-generating capacity of strips cut at various angles from their aortae. The greatest tension produced by strips of aortae of either group was generated by strips cut perpendicular to the long axis of the artery. Strips of aortae from either group produced less tension if cut at different angles with respect to the circumferential axis. Cut at any angle, the strips of aorta from DOCA-hypertensive rats produced less tension than those from control rats at the same preload. In length-active stress studies, stress developed by strips cut from DOCA-hypertensive rats was less than that of control rats throughout the entire range of resting lengths. Stereologic analysis of histologic sections of aortae demonstrated a decrease in the volume fraction of smooth muscle in the media of aortic wall. No differences between the carotid arteries of the same DOCA-hypertensive and normotensive rats were found in the proportion of water (by weight) in their walls.

The mechanism of action of barium ion on the aortic smooth muscle of the normal rat was investigated using in vitro calcium-depleted aortic strips. Aortic strips were depleted of calcium by repeated exposure to norepinephrine in a calcium-free bathing solution. Although calcium depletion abrogated the response of strips to catecholamines and depolarizing agents, the response to barium chloride remained quantitatively intact. The calcium influx blocker D 600 prevented the contractile response to barium but not to catecholamines, whereas phentolamine prevented the response to catecholamines but not barium. The strip response to barium was depressed by a twofold increase in extracellular magnesium concentration whether the strip was intact or calcium depleted. Although increased concentrations of calcium in the extracellular medium inhibited the contractile response to potassium ion, increases in barium merely potentiated the potassium contracture. These findings indicate that barium produces its contractile effect on vascular smooth muscle by a direct intracellular interaction with the contractile or regulatory proteins. Barium enters these cells via calcium influx channels and is probably not sequestered in a physiologically releasable pool. Unlike calcium, barium does not stabilize the smooth muscle sarcolemma when present in high concentration.