Full Text HL-94-014


NIH GUIDE, Volume 23, Number 17, May 6, 1994

RFA:  HL-94-014

P.T. 34

  Cardiovascular Diseases 

National Heart, Lung, and Blood Institute

Letter of Intent Receipt Date:  August 1, 1994
Application Receipt Date:  September 13, 1994


The Division of Heart and Vascular Diseases invites research grant
applications for up to four years of support for research into breast
cancer angiogenesis.  The objective of this Request for Application
is to encourage vascular biologists to apply their knowledge and
skills to elucidate the mechanisms where by breast tumor cells
stimulate angiogenesis and control the structure and function of the
tumor blood vessels.  The ultimate goal is to identify strategies
that offer possibilities for treating breast cancer by inhibiting the
vascularization of tumors.


The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This RFA,
Angiogenesis in Breast Cancer, is related to the priority area of
cancer.  Potential applicants may obtain a copy of "Healthy People
2000" (Full Report:  Stock No. 017-001-00474-0 or Summary Report:
Stock No. 017-001-00473-1) through the Superintendent of Documents,
Government Printing Office, Washington, DC 20402-9325 (202-783-3238).

Trans-NIH Breast Cancer Collaborative Effort

This RFA is part of the activities to be initiated by the National
Institutes of Health (NIH) to advance knowledge regarding the
etiology, treatment, and prevention of breast cancer.


Applications may be submitted by domestic and foreign for-profit and
non-profit organizations, public and private, such as universities,
colleges, hospitals, laboratories, units of State or local
governments, and eligible agencies of the Federal government.
Applications from minority individuals and women are encouraged.


This RFA will use the NIH individual research project grant (R01)
mechanism of support.  Responsibility for the planning, direction,
and execution of the proposed project will be solely that of the
applicant.  The total project period for applications submitted in
response to this RFA may not exceed four years.  This RFA is a
one-time solicitation.  Future unsolicited competing continuation
applications will compete with investigator-initiated applications
and be reviewed according to the customary peer review procedures.


Approximately $1.5 million in total costs will be provided for the
first year of support for the entire program.  It is anticipated that
no more than eight grants will be awarded under this program.  This
level of support is dependent upon the receipt of a sufficient number
of applications of high scientific merit.  Although this program is
provided for in the financial plan of the NHLBI, awards pursuant to
this RFA are contingent upon the availability of funds for this
purpose.  Administrative adjustments in project period and/or amount
of support may be required at the time of the award.

Since a variety of approaches would represent valid responses to this
RFA, it is anticipated that there will be a range of costs among
individual grants awarded.



The average U.S. mortality rate for breast cancer is 27.5 per hundred
thousand women.  Approximately 46,000 women died of breast cancer in
1993.  Thus, despite vigorous efforts to diagnose, treat and prevent
this disease, it remains a leading cause of death among women.  In
order to develop new approaches to the treatment of this disease,
further knowledge is needed of the basic biology underlying the
etiology and progression of the disease.  The orientation of this RFA
is on the mechanisms whereby cancer tumor cells promote angiogenesis
as a means of providing nutrients to the tumor cells and a conduit
for escape of tumor cells into the circulation.

Recent studies have shown that vascularization is significantly
higher in node-positive breast tumors than in node-negative tumors,
and that a high degree of vascularization is generally associated
with a poorer prognosis.  This  finding has led to the conclusion
that inhibition of angiogenesis offers a possible therapeutic
modality for a subset of patients.  Circulation in a tumor has both
favorable and detrimental aspects.  The existence of blood vessels
provides for the delivery of antitumor therapeutic agents but at the
same time it provides an escape route for metastases.

In a growing tumor, pre-existing host blood vessels become
incorporated into the tumor tissue.  These vessels, however,are
altered; venules are tortuous, elongated and dilated.  New blood
vessel growth occurs from these venules or from venules in the host
tissue adjacent to the invasion front.  Some of the original host
vessels in the tumor disintegrate, are obstructed or are compressed.
Of the remaining vessels, arteries seem to become permanently dilated
and resistant to the invasive and destructive growth of tumor cells.
The newly formed vessels randomly fuse either with arterioles or
venules and create a microcirculation that has many characteristics
not found in the microcirculation of normal tissues.  These
characteristics include abnormal morphology,absence of vasomotion,
increased fragility, and increased permeability.  Regions of a tumor
may be avascular or poorly vascularized and it is unclear why
angiogenesis fails in those regions.

A fundamental question that remains unanswered is how angiogenesis is
initiated in an avascular tumor.  In the heart, the stimulus is
thought to be ischemia and the vascular response is almost immediate.
Animal models subjected to embolization showed evidence of
angiogenesis in three days.  Tumors can remain avascular and survive
by growth at the periphery and necrosis at the center for several
years before angiogenesis begins.  On the other hand, some breast
tumors metastasize before they are clinically detectable and it is
not clear whether or not angiogenesis is a very early event in these
cases.  Investigations into the process by which tumor cells may
switch to the angiogenic phenotype include the following mechanisms:
(1) basic fibroblast growth factor (bFGF) normally confined within
cells or bound in the extracellular matrix is reported to be released
when tumor cells become angiogenic and (2) angiogenic inhibitors
secreted by cells decrease with the onset of angiogenesis presumably
due to a loss of a tumor suppressor.  The mechanism may be tumor
specific, and it is important to know whether these or other
mechanisms stimulate angiogenesis in breast cancer.

The first apparent response to an angiogenic stimulus from a tumor is
thought to be dissolution of the basement membrane surrounding a
pre-existing vessel, usually a post-capillary venule.  Endothelial
cells are then believed to migrate toward the tumor.  The leading
endothelial cells do not appear to divide, whereas those behind do.
After elongation of immature vessels, lumen formation occurs along
with branching and formation of vascular loops to produce a vascular
network.  Finally, capillaries become surrounded by basement membrane
components and, in most cases, pericytes.  However, as described in
further detail below, these processes differ from those seen in
vascularization of normal tissue.

The question of how these events are effected is complex but it is
clearly the result of interactions, either direct or indirect,
between tumor cells and endothelial cells.  Much progress has been
made in identifying factors that contribute to tumor angiogenesis.
In fact, more than a dozen purified molecules have been shown to be
angiogenic; most of them are polypeptides, but lipids, nucleotides,
copper, selenium, and one vitamin have also been shown to have
angiogenic properties.  The more difficult task is to now develop an
understanding of the cascade of events that result in vascularization
of a breast tumor and to identify specific factors and their precise
roles in that process.

Some of these factors are angiogenic in vivo but do not have
measurable effects on cultured cells, probably because the effect
requires the presence of other cells, such as macrophages that
produce endothelial activating factors, or because the factor is
normally stored in a cell or the extracellular matrix and is only
released under certain conditions.  Angiogenin is one factor that has
no effect on endothelial cell cultures.  Additionally, it is produced
in adult rat liver and is found in normal bovine and human plasma.
Thus, its activity as an angiogenic factor seems to be expressed only
in special circumstances when a receptor is presented on the
endothelial cell surface.

Some factors such as angiotropin, fibrin, and tumor necrosis
factor-alpha (TNF-alpha) stimulate endothelial cells to migrate, but
not to proliferate.  Others, such as basic fibroblast growth factor
(bFGF) and vascular endothelial growth factor (VEGF) are both
mitogenic and chemotactic.  Mitogenic factors can be produced by
macrophages, monocytes, platelets, and lymphocytes as well as tumor
cells.  Heparin-binding growth factors (HBGFs), including basic and
acidic FGFs, do not have signal peptides, yet appear to be released
into the subendothelial extracellular matrix and stored there bound
to heparin sulfate.  Degradation of the extracellular matrix is, in
itself, a complex process involving T and B lymphocytes, platelets,
granulocytes, macrophages, mast cells, and endothelial cells, as well
as compounds such as thrombin, and plasminogen activator (PA).  PA
inhibitors (PAIs) are also present.  Degradation appears to be under
the control of tumor cells.  A further step needed for the potent
mitogens to interact with endothelial cells is the expression of
receptors on those cells.  How all of these steps are integrated is
not well understood.

Ischemia/hypoxia is reported to be a stimulus for angiogenesis.
Recent studies have shown that VEGF is highly expressed in human
gliomas around the periphery of necrotic cords and that the VEGF
receptor is expressed only on endothelial cells.  In culture, a
glioma cell line was shown to express VEGF mRNA in response to
hypoxia.  Upon reoxygenation, mRNA levels returned to control levels.
Since VEGF expression is also influenced by estrogen, there may be a
particular relevance to breast cancer.  Other participants in the
angiogenic process are circulating cells recruited to tumor sites.
These include neutrophils, monocytes, macrophages, lymphocytes,
eosinophils, basophils, and mast cells.  Their specific roles are
only partially understood.  Of these cells, only macrophages appear
to have the ability to stimulate angiogenesis alone.  Mast cells may
play a role in the initiation of angiogenesis but this possibility
remains to be elucidated.  Heparin released from mast cells is
thought to have a role in potentiating angiogenesis but not to be
able act alone.  Moreover, in other circumstances, heparin appears to
be antiangiogenic.

It is important to note that the roles of angiogenic factors in tumor
vascularization may differ from those in wound healing.  There are
important distinctions between the structure and function of tumor
versus normal endothelium.  Thus, once the process of angiogenesis is
initiated, tumor cell-endothelial cell interactions become
determinants of the growth, morphology, and function of the
developing vessels.  For example, normal capillaries are often
surrounded by pericytes, whereas these may be reduced in number or
absent from tumor capillaries.  Pericytes are thought to be involved
in the maturation of vessels and the inhibition of endothelial
proliferation.  The combined activity of endothelial cells and
pericytes is thought to result in the elaboration of TGF-beta, an
inhibitor of endothelial cell proliferation. However, under certain
circumstances, TGF-beta can also promote angiogenesis.  Additionally,
the extracellular matrix of tumor vessels is different.  The basement
membrane surrounding the vessels is reduced, there are higher
concentrations of hyaluronic acid and lower concentrations of
sulfated proteoglycans.  The tumor vessels have increased
permeability, which may be due in part to the reduced basement
membrane, in part to increased fluid pressure in tumors and in part
to the elaboration of permeability factors, as well as to
modification or reduction of cell adhesion molecules.  In addition,
the increased permeability of the tumor vasculature may be due to
stimulation of nitric oxide release by FGF.  All of these features
would facilitate entry of tumor cells into the lumen in a manner not
possible for normal blood vessels.

An alternative source of angiogenic factors is the stroma, which is
specific to the breast and contains a variety of cells including
fibroblasts, macrophages and adipocytes. The stroma functions as a
source and storage site for cytokines and growth factors.  The
possibility that the stroma is abnormal in tumorogenesis has yet to
be investigated.

Although recent progress has led to the accumulation of much
knowledge on the subject of angiogenesis, that information has yet to
be synthesized in a manner that would allow the rational
consideration of new treatment modalities. Moreover, that knowledge
has been developed from studies of normal tissues, bone marrow, and
many different tumors.  The purpose of this initiative is to focus on
breast tumors and on the specific mechanisms of stimulation and
inhibition of angiogenesis in those tissues.  The powerful tools of
molecular biology and immunology combined with animal models and
possibly cell lines provide the means to increase knowledge in this
area.  Lastly, although the lymphatic system is known to play a
critical role in the process of metastasis, it remains unclear
whether there are lymphatics within the breast tumor and how the
tumor vasculature relates to the host lymphatic system.

Research Topics

The following suggestions are given only as examples.  They are not
intended to represent research directions to which the NHLBI would
give special emphasis.  Investigators are urged to utilize their own
knowledge of the subject in preparing a response to this RFA.

o  changes in tumor cell gene expression that elicit angiogenesis in
breast tumors

o  mechanisms underlying the expression of receptors for angiogenic
factors on endothelial cells

o  interactions between tumor cells, circulating cells, and
endothelium in  breast tumors

o  role of the stroma in promotion of angiogenesis

o  mechanisms that direct endothelial cell migration into the tumor

o  molecular regulation of angiogenic factor production by breast

o  mechanisms of control of gene expression in endothelial cells
residing in breast tumors

o  relative roles of tumor cells and altered endothelial cells in the
modification of extracellular matrix associated with tumor

o  role of hormones in the angiogenic process in breast cancer

o  endogenous inhibitors of angiogenesis

o  the relative roles of specific oncogenes and tumor suppressor
genes in modulating the process of metastasis

o  the relationship of the tumor vasculature and possible lymphatics
to the host blood vessels and lymphatic system

Investigators should be aware that NIH requires applicants to comply,
where feasible and appropriate, to the inclusion of minorities and
women in study populations.


Although multidisciplinary approaches are encouraged, it is not the
intent of this RFA to solicit applications for large studies that
would encompass a variety of independent projects, e.g., program
projects.  This program will not support clinical trials or large
epidemiological studies.  In general, funds will not be provided for
the purchase and installation of expensive, equipment.  Awards under
this RFA to foreign institutions will be made only for research of
very unusual merit, need and promise, and in accordance with PHS
policy governing such awards.

Upon initiation of the program, the Division of Heart and Vascular
Diseases will sponsor periodic meetings to encourage the exchange of
information among grantees and to stimulate collaboration.
Applicants should include their budget request funds for travel to an
annual one-day meeting, most likely to be held in Bethesda, Maryland,
and should also include a statement indicating their willingness to
participate in these meetings.



It is the policy of the NIH that women and members of minority groups
and their subpopulations must be included in all NIH supported
biomedical and behavioral research projects involving human subjects,
unless a clear and compelling rationale and justification is provided
that inclusion is inappropriate with respect to the health of the
subjects or the purpose of the research.  This new policy results
from the NIH Revitalization Act of 1993 (Section 492B of Public Law
103-43) and supersedes and strengthens the previous policies
(Concerning the Inclusion of Women in Study Populations, and
Concerning the Inclusion of Minorities in Study Populations), which
have been in effect since 1990. The new policy contains some
provisions that are substantially different from the 1990 policies.

All investigators proposing research involving human subjects should
read the "NIH Guidelines For Inclusion of Women and Minorities as
Subjects in Clinical Research," which have been published in the
Federal Register of March 9, 1994 (FR 59 11146-11151) and reprinted
in the NIH Guide for Grants and Contracts, Volume 23, Number 11,
March 18, 1994.

Investigators also may obtain copies of the policy from the program
staff listed under INQUIRIES.  Program staff may also provide
additional relevant information concerning the policy.


Prospective applicants are asked to submit, by August 1, 1994, a
letter of intent that includes a descriptive title of the proposed
research, the name, address, and telephone number of the principal
investigator, the identities of other key personnel and participating
institutions, and the number and title of the RFA in response to
which the application may be submitted.

Although a letter of intent is not required, is not binding, and does
not enter into the review of subsequent applications, the information
that it contains is helpful in planning for the review of
applications.  It allows NHLBI staff to estimate the potential review
workload and to avoid conflict of interest in the review.

The letter of intent is to be sent to:

C. James Scheirer, Ph.D.
Division of Extramural Affairs
National Heart, Lung and Blood Institute
Westwood Building, Room 557
Bethesda, MD  20892


The research grant application for PHS 398 (rev. 9/91) is to be used
in applying for these grants.  These forms are available at most
institutional offices of sponsored research and may be obtained from
the Office of Grants Information, Division of Research Grants,
National Institutes of Health, Westwood Building, Room 449, Bethesda,
MD 20892, telephone (301-710-0267).

The RFA label in the PHS 398 application form must be affixed to the
bottom of the face page of the application.  Failure to use this
label could delay processing of the application such that it may not
reach the review committee in time for review.  In addition, to
identify the application as a response to this RFA, Check "YES",
enter the title "Angiogenesis in Breast Cancer", and the RFA number
HL-94-014 on Line 2a of the face page of the application.

Send or deliver a signed, typewritten original of the application,
including the checklist, and three signed photocopies, in one package

Division of Research Grants
National Institutes of Health
Westwood Building, Room 240
Bethesda, MD  20892**

Send two additional copies of the application to the Chief, Centers
and Special Projects Section, at the address listed under LETTER OF
INTENT.  It is important to send these two copies at the same time as
the original and three copies are sent to the Division of Research
Grants; otherwise, the NHLBI cannot guarantee that the application
will be reviewed in competition for this RFA.

Applicants from institutions that have a General Clinical Research
Center (GCRC) funded by the NIH National Center for Research
Resources may wish to identify the GCRC as a resource for conducting
the proposed research.  If so, a letter of agreement from either the
GCRC program director or principal investigator could be included
with the application.

Applications must be received by September 13, 1994.  If an
application is received after that date, it will be returned to the
applicant.  The Division of Research Grants (DRG) will not accept any
application in response to this RFA that is essentially the same as
one currently pending initial review, unless the applicant withdraws
the pending application.  The DRG will not accept any application
that is essentially the same as one already reviewed.  This does not
preclude the submission of substantial revisions of applications
already reviewed, but such applications must include an introduction
addressing the previous critique.


Letter of Intent Receipt Date:     August 1, 1994
Application Receipt Date:          September 13, 1994
Review by NHLBI Advisory Council:  February 1995
Anticipated Award Date:            May 1995


Upon receipt, applications will be reviewed by NIH staff for
completeness and responsiveness.  Incomplete applications will be
returned without further consideration.  If the application is
complete but not responsive to the RFA, NHLBI staff will contact the
applicant to determine whether to return the application or to submit
it for review in competition with unsolicited applications at the
next review cycle.

Applications may be triaged by an NHLBI peer review group on the
basis of relative competitiveness.  The NIH will withdraw from
further competition those applications judged to be non-competitive
for award and notify the applicant Principal Investigator and
institutional official.  Those applications judged to be competitive
will undergo further scientific merit review.  Those applications
that are complete and responsive will be evaluated in accordance with
the criteria stated below for scientific/technical merit by an
appropriate peer review group convened by the NHLBI.  The second
level of review will be provided by the National Heart, Lung, Blood
Advisory Council.

Review Criteria

The review criteria are:

o  the novelty, originality, and feasibility of the approach and the
adequacy of the experimental design

o  the competence of the principal investigator and collaborators to
accomplish the proposed research, and the commitment and time they
will devote to the project

o  the suitability of the facilities to perform the proposed
research, including laboratories, instrumentation, and data
management systems

o  the appropriateness of the requested budget and duration for the
proposed research

o  the adequacy of plans for interaction and communication of
information and concepts among investigators involved in
collaborative studies


Applications must fulfill all the eligibility criteria in order to be
considered for funding.  The most important criterion in selecting
awardees will be  scientific merit, as reflected in the priority
score.  However, factors such as program balance and available funds
may also enter into the selection of meritorious applications.

The anticipated date of award is May 1995.


Written and telephone inquiries concerning this RFA are encouraged.
The opportunity to clarify any issues or questions from potential
applicants is welcome.

Inquiries regarding programmatic issues may be directed to:

Dr. Constance Weinstein
Division of Heart and Vascular Diseases
National Heart, Lung, and Blood Institute
Federal Building, Room 3C06
Bethesda, MD  20892
Telephone:  (301) 496-1081
FAX:  (301) 480-6282

Inquiries regarding fiscal and administrative may be directed to:

Mr. William Darby
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 4A11
Bethesda, MD  20892
Telephone:  (301) 594-7458
FAX:  (301) 594-7492


This program is described in the Catalog of Federal Domestic
Assistance number 93.837, Heart and Vascular Diseases.  Awards will
be made under the authority of the Public Health Service Act, Section
301 (42 USC 241) and administered under PHS grants policies and
Federal regulations, most specifically 42 CFR Part 52 and 45 CFR Part
74.  This program is not subject to the intergovernmental review
requirements of Executive Order 12372, or to Health Systems Agency

The Public Health Service (PHS) strongly encourages all grant
recipients to provide a smoke-free workplace and promote the non-use
of all tobacco products.  This is consistent with the PHS mission to
protect and advance the physical and mental health of the American


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